Course title: Techniques in Microbiology Course description: The course is designed to provide students a basic knowledge, principles and

applications of basic and advanced laboratory techniques in microbiology which are required to implement relevant community based research projects and of course, for the diagnosis of infectious diseases.

Course objectives:- The course attempts to Provide basic principles and skills on Microbiological techniques for processing specimens for the isolation and identification of bacterial, fungal, and parasitic organisms commonly encountered in infectious processes. The students will also perform basic instruction and practice in appropriate techniques for antimicrobial susceptibility testing.

Section I: Basic Microbiological techniques Section: Advanced methods in Microbiology 1. INTRODUCTION Microbiological investigations are important in the diagnosis, treatment, prognosis and surveillance of infectious diseases. According to an old Chinese proverb: Tell me and I will forget. Show me and I might remember. Involve me and I will understand. These words convey our basic philosophy that it is experiences in the microbiology laboratory and the scientific method that help develop students critical thinking and creativity and that increase their appreciation of the mechanisms by which microbiologists analyze information. The laboratory accomplishes this by having students become intensely and personally involved in the knowledge they acquire. 2. SAFETY PROCEDURES In the microbiology laboratory you will be handling billions of living bacterial cells and you must follow certain rules to protect yourself and those around you. The bacteria that you will be working with ability of disease causing under normal circumstances, all bacteria are potentially harmful and should be treated with care. Other sources of harm common to any laboratory include flames from Bunsen burners and broken glass. The following is a set of rules that you should read and observe. Personal care: 1. All materials and clothes other than those needed for the laboratory are to be kept away from the work area. 2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be worn outside of the laboratory. 3. Clean the lab table before and after lab with the disinfectant solution provided. 4. Wash hands before leaving lab. 5. Because organisms used in this class are potentially pathogenic, aseptic technique must be observed at all times. 6. NO eating, drinking, application of cosmetics or smoking is allowed. 7. Long hair should be tied back while in lab. 8. Microscopes and other instruments are to be cared for as directed by the instructor. 9. Visitors are not allowed in the lab. 10. Doors and windows are to be kept closed at all times. 11. For the best lab experience, read labs before coming to class. Make notes as necessary. Wait for a laboratory introduction by the instructor before starting work. 12. Individuals can be exposed in various ways to laboratory acquired infections in microbiology laboratories. These involves: Rubbing the eyes or nose with contaminated hands. Inhaling aerosols produced during centrifugation, vortexing or spills of liquid cultures. Accidentally ingesting micro-organisms by putting pens or fingers in the mouth. Suffering percutaneous inoculation, i.e. being punctured by a needle stick.

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Safe disposal of specimens: 1. Disposable GLOVES should be used. 2. Any item contaminated with bacteria or body fluids must be disposed of properly. 3. Disposable items are to be placed in the BIOHAZARD container. 4. Reusable items are to be placed in the designated area for autoclaving prior to cleaning. 5. Sharp materials are to be disposed of in an appropriate container. 6. Reusable items should have all tape and marks removed by the student before being autoclaved. 7. Sterilization techniques will involve the use of bacticinerators that are fire and burn hazards. Bacticinerators reach an internal temperature of 850 oC or 1500o F, Keep all combustibles away from the bacticinerators. Emergency precautions 1. Cuts and scratches must be covered with Band-Aids. 2. It is the responsibility of the student to know the location and use of all safety equipment in the lab (eyewash, fire extinguisher, etc.) 3. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructor immediately. 4. In case of accident where cultures are spilled or dropped: 1. Cover the spill with disinfectant soaked paper towels. 2. Report the accident. 3. Clean up glass with dust pan and hand broom provided. Never brush up broken glass with your hands. 5. Discard paper towels in BIOHAZARD bag. 3. DEMONSTRATIONS Demonstration of laboratory equipments and their use Wire loop /straight wire, Incubator, Cover slide, Petri dish, Sprit lamp/Bunsen burner, Microscope, Test tubes, Flasks, Candle jar, Autoclave, Centrifuge, Safety cabinet, Hot air oven, CO2 incubator, Colony counter, Micropipette, Spectrophotometer .etc Demonstration of laboratory request forms Bacteriology Parasitology Urine analysis Hematology Serology Chemistry Blood bank

Demonstration of different sample containers and required materials for sample collection 4. Sputum cup Cover slide Stool cup, Syringe, needle Glass Slide Wide mouthed bottle Vaginal speculum Blood culture bottle Blood lancet Sterile test tube with cotton swab Citrated bottle Clean test tube Scalpels Forceps. Etc

COLLECTION, TRANSPORTATION AND PRESERVATION OF MICROBIOLOGICAL SPECIMENS

If Pathogens are to be isolated successfully and thus the patient be treated rationally, the type of specimen, its collection, and the time and method of its dispatch to the laboratory must be correct. There must be adequate information about the patients condition and antimicrobial treatment. Type of Specimen: Blood Sputum Wound Stool CSF Urine Aspirates Skin Scrapings

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Collecting samples aseptically The laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen. Proper collection of an appropriate clinical specimen and transported in the right environment to the laboratory is the first step in obtaining an accurate laboratory diagnosis of an infectious disease. Basic requirements before sending specimens for culture Following measures should be taken before submitting specimens for culture to the clinical laboratory. 1. 2. 3. 4. 5. 6. 7. 8. Collect the specimen before the administration of antimicrobial agents The specimen should be collected aseptically to prevent the contamination of the specimen with superficial organisms or normal flora of the body Make sure that the container is not contaminated, and is leak proof. Follow strict aseptic techniques throughout the procedure. Make sure that the specimen is representative of the infectious process (e.g. early morning quality sputum sample) and is adequate in quantity. Label the container appropriately and complete the request form. The specimen should be transported immediately to the laboratory. Information regarding the patient, the specimen, collection time and date, clinical history, symptoms and diagnosis, antimicrobial therapy (or use of any other chemotherapeutic agents) and any suspected organisms is essential for the optimal and appropriate processing of the specimen.

Blood specimen: 1. 2. 3. 4. After placing a tourniquet on the patient's arm, select a suitable vein. Cleanse the area with 2% iodine followed by 70% alcohol, in circular motion from the center to outside. Without touching the site of the venipuncture with the fingers, draw 5-10mL of blood into a sterile syringe. First clean the blood culture bottle stoppers with alcohol then inoculate 5-10mL of the specimen into each of two culture bottles. Gently invert the bottles several times to insure thorough mixing.

Points that should be communicated to the clinicians before blood culture collection 1. 2. 3. Collect blood during the early stages of disease, during peak of fever since the number of bacteria in blood is higher in the acute and early stages of disease. Blood cultures should be sent prior to antibiotic administration. Three blood cultures sets are recommended at one hour intervals to obtain 99% positivity.

Small children usually have higher number of bacteria in their blood as compared to adults and hence lesser volume (3-8 mL) is required for culture. Cerebrospinal Fluid (CSF) Examination of CSF is an essential step in the diagnosis of meningitis 1. Collect CSF in a screw capped sterile container and not in an injection vial with cotton plug. 2. Almost 3-5 ml of CSF should be collected for biochemical, immunological and microscopic examination. 3. Do not delay in transportation and laboratory investigations. 4. Transport in a transport medium if delay in processing is unavoidable. 5. CSF is a precious specimen, handle it carefully and economically. It may not be possible to get a repeat specimen. Sputum: 1. Select a good wide-mouthed sputum sterile container, which is preferably disposable, made of clear thin plastic, unbreakable and leak proof material. 2. Three early morning sputum specimens are recommended for diagnosis of tuberculosis 3. Explain to the patient to rinse his/her mouth with plain water before bringing up the sputum. 4. Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in the sputum container by bringing it closer to the mouth. 5. Make sure the sputum sample is of good quality. A good sputum sample is thick, purulent and sufficient in amount (2-3 ml). 6. Do not submit saliva to the laboratory as it would lead to erroneous results.

Urine: The lower part of the urethra and the genitalia are normally colonized by bacteria, therefore proper aseptic techniques should be used to prevent contamination of urine with the perianal flora.

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1.

Collect "clean catch-mid-stream" specimen (Early morning specimen is recommended, if this is not possible, then specimen should be collected two hours after last micturition). 2. Specimens should be transported to the laboratory within one hour for bacteriological examination. If delay is expected in transportation collect specimen in 1 % boric acid and containers brought to laboratory within 4-6 hours. 3. Transportation should be in cooling box (2-8C) except when transport time is very short. Male 1. 2. 3. Female If not circumcised, draw back the fore skin. Discard initial few ml of the urine. Collect the mid-portion of the urine into container, and pass the excess in the toilet.

1. Wash the area around the urethra by means of water.

2. 3. Squat over the toilet and separate the labia with one hand. Discard initial few ml in the toilet and collect the mid-portion of urine in the container.

Indwelling Catheter urine specimen: 1. 2. 3. Clean the catheter collection port with 70% alcohol wipe. Using sterile technique, puncture the collection port with a needle attached to the syringe. Aspirate the urine and place in sterile container.

Wound Specimens: 1. Pus from an abscess is best collected at the time the abscess is incised and drained, or after it has ruptured naturally. 2. When collecting pus from wounds, or other sites, special care should be taken to avoid contaminating the specimen with commensal organisms from the skin. 3. As far as possible, a specimen from a wound should be collected before an antiseptic dressing is applied. 4. Use a sterile cotton-wool swab to collect a sample from the infected site. Immerse the swab in a container of Amies transport medium. Aspirates: Aspirated specimens, fluids, tissues are good quality specimens that are preferable than specimens collected on swabs. Mention special instructions or specific tests to be done on requisition slip.(e.g. for fungal culture or AFB culture or Nocardia or any other fastidious bacteria) Try to follow the universal precautions for specimen collection if collecting with swab. Swabs are unacceptable for anaerobic culture and TB cultures. Try to transport the aspirate as early as possible to the laboratory in order to prevent contamination and also for maximum yield of aerobic as well as anaerobic bacteria.

Stool Specimens: For bacteriologic examination, fresh stool specimen is recommended. Specimen should be collected in a clean wide mouthed plastic bottle. A wooden stick to transfer the specimen should be provided. For bacterial culture also, fresh stool must be submitted, if delay in transportation is expected to be more than 2 hours, specimen should be submitted in Cary Blair Medium.

Time of Collection Specimens such as urine and sputum are best collected soon after a patient wakes when organisms have had the opportunity to multiply over several hours. Blood for culture is usually best collected when patients temperature begins to rise.

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The time of collection for most other specimens will depend on the condition of the patient, and the times agreed between the medical, nursing and laboratory staff for the delivery of specimens to the laboratory. Every effort must be made to collect specimens for microbiological investigation before antimicrobial treatment is started.

Labeling of Specimens Each specimen must be clearly labeled with the date and time of collection, and the patients name, number, ward or outpatient department. Slides with one end frosted should be used for making smears so that a lead pencil can be used to label the slides clearly. Each specimen must be accompanied by a correctly completed request from which details: The patients name, age, gender, number and ward or OPD. Type of specimen, and the date and time of its collection. Investigation required. Clinical note giving details of the patients illness, suspected diagnosis and any antimicrobial treatment that may have been started at home or in the hospital. Transportation and Preservation of Microbiological Specimens In general, specimens for microbiological investigations should be delivered to the laboratory as soon as possible. This will help to ensure that pathogens are viable when they reach the laboratory. Amies transport medium is widely used in the viability of pathogens such as Neisseria gonorrhoeae. Cary-Blair medium is used as a transport medium for faeces that may contain Salmonella, Shigella or Vibrio species. Preservatives include boric acid for urine, cetylpyridinium chloride for sputum for M.tuberculosis. In unpreserved specimens, refrigeration at 4-10%c can help to preserve cells and reduce the multiplication of commensals. Refrigeration is not recommended for specimens for the isolation of Haemophilus, S.pneumoniae or Neisseria species. Storage of bacterial culture Freezing: 1. Grow cells to late log or early stationary phase under optimal conditions on the medium of choice. 2. If broth culture is used, centrifuge to harvest the cells. Pour off the supernatant. 3. Re-suspend the cells in a smaller volume of the same growth medium, and add an equal amount of 20% (vol/vol) glycerol. 4. If cells were grown on agar slants, wash off the growth with the appropriate broth and add an equal amount of 20% glycerol. 5. Aliquot into small plastic vials and freeze. The lower the temperature, the longer the cells will survive. Liquid nitrogen storage is the most ideal. , Glycerol storage of bacterial cultures 1. To 0.85 ml [3 ml] of bacterial culture, add 0.15 ml [0.53 ml] of sterile glycerol (sterilized by autoclave, final glycerol concentration: 15%, v/v). 2. Vortex the culture to ensure that the glycerol is evenly dispersed. 3. Transfer the culture to a labeled storage tube equipped with a screw cap. 4. Freeze the culture in dry ice or in liquid nitrogen, and then transfer the tube to 70 C for long-term storage. 5. MICROSCOPY Introduction A microscope is an essential tool for any microbiology laboratory. The commonly used microscopes in diagnostic work are the compound light microscope, dark field, phase contrast and fluorescent microscope. The following are important parts of the compound microscope and their function: 1. Note that its working parts are set into a sturdy frame consisting of a base for support and an arm for carrying it. 2. Observe that a flat platform, or stage as it is called, extends between the upper lens system and the lower set of devices for providing light. The stage has a hole in the center that permits light from below to pass upward into the lenses above. The object to be viewed is positioned on the stage over this opening so that it is brightly illuminated

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3. 4. 5.

6.
7. 8.

from below. Microscopes are equipped with a mechanical stage, once the specimen is placed into position it no longer needs adjustment by directly touching it. The light source is at the base. Most microscopes have a built in illuminator. The condenser contains lenses that collect and concentrate the light, directing it upward through any object on the stage. Above the stage, attached to the arm, a tube holds the objective lenses through which the object is viewed. The lower end of the tube is fitted with a rotating nosepiece holding three or four objective lenses. As the nosepiece is rotated, any chosen objective can be brought into position above the stage opening. The upper end of the tube holds the ocular lens, or eyepiece. The rotating nosepiece can be raised or lowered by means of coarse and fine adjustment knobs. These are located below the stage. Bring the lens safely down first with the coarse knob, then, while looking through the ocular, turn the fine knob to raise the lens until you have a clear view of the subject. The total magnification achieved with the microscope depends on the combination of the ocular and objective lens used.

OTHER TYPES OF MICROSCOPES

1. Dark field Microscope: The same lenses used in bright-field microscope are used in dark field microscope.
However, special condenser is used in this microscope. Resolving power is 0.02um. This allow to see extremely thin bacteria such as ( Treponema pallidum : etiologic agent of syphilis )

2. Phase - contrast microscope: This microscope enables the internal details of microbe to be examined. The
annular rings use in condenser and the objective lenses. This microscope gives three dimensional image (3D).

3. Fluorescence Microscope: This microscope use fluorescent dyes (stain). The microscope use high pressure
mercury halogen or xenon vapor.

4. Electron Microscope: Unlike other forms of microscope, magnetic coils (rather than lenses) this microscope
used beam of electron instead of light. Samples are usually stained by metal ions to create contrast. There are two types of electron microscope: Transmission electron microscope : In which electrons pass directly through the specimen , magnification of it is ((10000 100000 X)) and image in this type not give (3D) picture Scanning electron microscope : magnify ((1000-10000X)) , the image I (3D) Demonstration of Microscopic Examination of Prepared Slides Now that you are familiar with the parts and mechanisms of the microscope, you are ready to learn how to focus and use it to study microorganisms. Materials: 1 microscope, 3 prepared stained bacterial slides, 1 tube immersion oil 1. 2. 3. 4. 5. 6. 7. Using both hands, remove the microscope from its cabinet and place it on your bench. Use one hand to grasp the arm of the microscope and support it from beneath with the other. To obtain a clear image, clean the ocular and the objective lenses before use. Use only lens paper to clean lenses. Never touch the lenses with your fingers. Place a slide of stained bacterial cells on the stage with the stain side up. Plug in the light source and turn it on. Adjust the condenser to a position close to the top of its travel. Move the slide around on the stage until you can see light shining through the stained material. Move the slide using the knobs located on the side of the stage. Grasp the nosepiece (not the objectives) and rotate it so that the 10X scanning objective is positioned over the slide. Watch the objective from the side and lower the nosepiece using the coarse focus knob (the larger one) until it reaches the bottom of its travel. The scanning objective and the slide are now as close together as possible. Focus on the stained material with the 10X scanning objective using the coarse focus knob by slowly raising the nosepiece. The stained material should make a sharp image, but individual bacterial cells will not be visible. Close the iris diaphragm to obtain good contrast. Best position is nearly closed.

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8.

9. 10.

11. 12. 13. 14. 15.

Once the stained material is in focus under the scanning objective, the coarse focus knob should not be used any more. The microscope should be approximately in focus with the high dry (40X) or oil immersion (100X) objective in position. A microscope that has all the objectives approximately in focus at the same point of travel is said to be parafocal. Any further focusing should be done with the fine focus (small knob). Rotate the high dry objective into position. Sharpen the image using the fine focus knob. Adjust the diaphragm as necessary. Rotate the high-dry objective out of position and place a single drop of immersion oil directly on the slide. Rotate the oil immersion objective into position. The tip of the objective should touch the oil so that there is not air between slide and objective. Use the fine focus knob to obtain a sharp image. You should be able to distinguish individual bacterial cells. To look at another slide, you should repeat the entire process. As soon as you are finished observing a slide that is to be saved, wipe the oil off the slide using lens paper. When you are finished using the microscope, take a small piece of lens paper folded double and wipe off all the lenses in the order (1) ocular, (2) scanning, (3) high-dry, (4) oil immersion. Be sure to remove all oil from the oil immersion objective. Be sure that the slide is removed. Position the 10 X-scanning objectives and raise the nosepiece. Return the microscope to its cabinet in the proper numbered location. View each of the three slides provided.

Results:

6. MICROSCOPIC EXAMINATION OF CLINICAL SPECIMENS Microscopically, the presence of bacterial pathogens can be detected by two ways: Unstained preparation Stained preparation Examination of Unstained Preparations The observation of unstained living organisms is required to determine whether the culture of bacteria belongs to a species that is motile or to identify different cells and parasites. Commonly used unstained preparations are: Wet mount Hanging drop KOH Wet mount preparation Objectives: To examine specimens and cultures for motile bacteria, capsulated yeast cells, identify different cells and parasites. Materials: - Slide - Cover slide - Applicator stick - Normal saline (0.85%w/v) - Microscope Procedure: - Place a drop of normal saline on the center of the slide and mix with a clinical specimen. OR - Place a small amount of emulsified specimen onto the microscope slide. - Cover with cover glass. - Examine using 40X objectives.. Result: Observe motility of the microorganism and look different cells and draw the diagram. Hanging drop Preparation Objective: To know whether an organism is motile or non-motile. Principle: This technique is meant for microscopic observation of living bacteria. The motility and binary fission may be seen using this technique.

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Materials required:

- Cavity /well slide - Cover glass - Applicator stick - Soft petroleum jelly - Microscope.

Procedure: Encircle the cover glass with a line streak of soft petroleum jelly using applicator stick. Place a drop of bacterial suspension on cover glass. Invert the well slide over the cover glass, allowing it to adhere to the petroleum jelly. Then quickly turn round the well slide so that the cover glass is uppermost. The drop will then be hanging from the cover glass in the center of the well slide. Examine using 10X and 40X objectives, respectively. Result: Look for motility of microorganisms.

Examination of stained preparations As you have discussed above, wet mounts of bacterial cultures can be very informative, but they have limitations. Bacteria bounce about in fluid suspensions, with Brownian movement or true motility, and are difficult to visualize sharply. We can see their shapes and appreciate their activity under a cover glass, but it is difficult to form a complete idea of their morphology. Because they are so small and have so little substance, they tend to be transparent, even when magnified is subdued light. The trick, then, is to find ways to still their motion and tag their structures with something that will make them more visible to the human eye. Many sophisticated ways of doing this are known, but the simplest is to smear out a bacterial suspension, fix the organisms to the slide, and then stain them with a visible dye. The stains are acidic, basic, or neutral in reactivity. Acidic or basic stains are used in bacteriologic work, primarily. The free ions of acidic dyes are anions (negatively charged) that combine with cations of a base in the stained cell to form a salt. Basic dyes possess cations (positively charged) that combine with an acid in the stained material to form a salt. Bacterial cells are rich in ribonucleic acid (contained in their abundant ribosomes) and therefore stain very well with basic dyes. Neutral stains are made by combining acid and basic dyes. They are most useful for staining complex cells of higher forms, for they permit differentiation of interior structures some of which are basic, some acidic. 5.2.1 Smear preparation The preparation of a smear is required for many laboratory procedures, including the Gram-stain. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium. Below are some guidelines for preparing a smear for a Gram-stain. Materials required: Clean glass slides Prepared cultures of Bacteria Inoculating loop Bacticinerator/ spirit lamp Laboratory marker

Procedure 1. Glass slides should be relatively clean and grease free. Slides that do not appear clean may be washed in soap and water and dried with a paper towel. 2. Work with one slide at a time. Sterilize an inoculating loop. Aseptically remove the lid from the water bottle and remove a loopful of water from the bottle. Return the lid to the bottle. 3. Tap the loopful of water onto the center of one of the labeled slides. 4. Sterilize the loop. 5. Obtain a slant culture of one of the organisms. Aseptically remove the lid. Insert the sterile loop into the tube being careful not to touch the lip of the tube. Touch the loop to the surface of the agar. DO NOT scrape or dig into the agar. Remove the loop and return the lid to the tube. 6. Mix the material on the loop in the drop of water on the appropriately labeled slide. Spread the drop over the surface of the slide making a uniform preparation of bacteria and water. The thinner the smear the quicker it will dry.

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7. 8. 9.

Allow the smear to air dry. Heat fix the slide by passing it 10 times over the top of the Bacticinerator. The slide is ready for staining. It may be stored until needed.

Staining Methods Staining helps to increase the refractive index of the organism, therefore staining is an important consideration in the observation and identification of microorganisms. Depends on the position of the coloring agent, stains can be basic, acidic and neutral. Basic stains:- are stains that contain the coloring agent in the base part and the acid part is colorless. Ex. Crystal violet, basic fuchsine and methylene blue Acidic stains:- are stains that contain the coloring agent in the acid part and the base part is colorless. Ex. eosin Neutral stains:- are those stains in which both the acid and base parts contain the coloring agent. Ex. Giemsa stain. Types of staining 1. Simple staining - use single stain or dye. 2. Differential staining use different more than one dyes. 3. Special staining stains special structure of the microorganism. Simple positive staining Objective: To show not only the presence of microorganism but also the nature of the cellular content in exudates.

Principle: The color giving substance gives color for the colorless microorganisms, so that the microorganism or its parts are stained by the dye. Materials required: - Slide - Applicator stick - Wire loop - Sprit lamp - Positive stain (methylene blue, - (1%) carblofuchsin (1%), - Gentian violet (1%). - Clean water - Microscope - Immersion oil Procedure: - Make the smear from direct clinical specimen or from culture and label it. - Allow the smear to air dry. - Fix the smear - Cover the smear with a positive stain and wait for 1min. - Wash off the stain with clean water. - Air-dry and examine under 100x objective. Result: Look for the presence or absence of microorganism. - identify the morphology of the mos (cocci, rods) - Identify the arrangement (in pairs, in clusters, in chains)

Simple Negative Staining Objective: To see capsulated microorganisms in C.S.F, like Cryptococcus neoformans. Principle: The color giving substance gives color for the background and the micro- organism remain unstained. -Slide - Cover glass - Applicator stick - Wire loop - Sprit lamp - Indian ink stain - Microscope.

Materials required:

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Procedure: - Put a specimen on the slide or bacterial suspension from culture. - A small quantity of Indian ink is added. - Mix - Cover with cover glass - Examine under microscope. Result: Look for retractile capsulated microorganism on the black background.

Grams staining Objective: To categorize bacteria as Gram positive and Gram negative and to observe morphology. Principle: Differences in Gram reaction between bacteria is thought to be due to differences in the permeability of the cell wall of Gram positive and Gram negative organisms during the staining process. Following staining with a triphenyl methane basic dye such as crystal violet and treatment with iodine, the dye-To dine complex is easily removed from the more permeable cell wall of Gram positive bacteria. Retention of crystal violet by Gram positive organisms may also be due in part to the more acidic protoplasm of these organisms binding to the basic dye. Materials required: - Slide - Applicator stick - Wire loop - Sprit lamp - Cotton/Gauze - Crystal violet stain - Lugols iodine - Aceton- alcohol - Neutral red/saffranin - Immersion oil - Microscope Procedure: Prepare a thin smear of the culture or specimen to be observed. Allow to air-dry and fix the smear. Cover the fixed smear with crystal violet for 1 min. Rinse with clean water and tip off all the water. Cover the smear with Lugols iodine for 1 min. Wash off the iodine with clean water. Add acetone-alcohol for 30 sec. Wash the smear immediately with clean water. Cover the smear with saffranin for 1-2 minutes. Rinse with clean water. Wipe the back of the slide and place in a draining rack for the smear to air-dry. Examine microscopically, first with the 40x objective and then with the oil immersion objective for white cells, bacteria and other structures. Results: - Gram- positive bacteria -------------Dark purple - Gram- negative bacteria ------------Pale to dark red. - Yeast cells ----------------------------Dark purple - Nuclei of pus cells ------------------Red - Epithelial cells -----------------------Pale red. Reporting Gram smears: The report should include the following information: Numbers of bacteria present (Many, Moderate, Few or scanty) Gram reaction of the bacteria (Gram +ve or Gram ve)

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NOTE: Gram-positive bacteria appear gram-negative if: The cell wall is damaged due to antibiotic therapy or excessive heat-fixation of the smear. The smear is over decolorized. The smear is prepared from old culture. The iodine used is too old. Gram-negative bacteria appear gram-positive if: The smear is too thick. Acid Fast /Ziehl-Neelsen/Staining Technique Objective: To stain mycobacterium species including M.tuberculosis, M.ulcerans and M.leprae and to differentiate from other microorganisms. Principle: The cell walls of certain bacteria contain long-chain fatty acids (mycolic acids), lending them the property of resistance to de-staining of basic dyes by acid-alcohol. Thus, they are called Acid Fast Bacilli (AFB). Mycobacterium, unlike most other bacteria, does not stain well by the Gram technique. They can however be stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in the mycobacterial cell wall. After staining, an acid decolorizing solution is applied. This removes the red dye from the background cells, tissue fibers and any organisms in the smear except mycobacteria which retain the dye. Following decolonization, the smear is counter stained with malachite green or methylene blue which stains the background material, providing a contrast colour against which the red AFB can be seen. Materials required: - Slide - Applicator stick - Wire loop - Sprit lamp - Cotton/Gauze - Carbol fuchsin - Acid- alcohol (1% or 3%) -Methylene blue or Malachite green. - Immersion oil - Microscope Procedure: - make a smear an air dry. - Fix the dried smear by heat or alcohol. - Cover the smear with carbol fuchsin stain. - Heat the stain until vapor just begins to rise and allow the heated stain to remain on the slide for 5 minutes. - Wash off the stain with clean water. - Cover the smear with 1% or 3% acid alcohol for 3 minutes. - Wash with clean water. - Cover the smear with methylene blue (malachite green) for 1 min. - Wash with clean water. - Wipe the back of the slide and let it air dry. - Examine the smear microscopically using oil-immersion objective. Result: AFB ------------ Red, straight or slightly curved rods, occurs singly or in small groups. Background material ---------------blue or green Cells ------------------------------blue or green Reporting of sputum smear: When any definite red bacilli are seen, report the smear as AFB positive and give an indication of the number of bacteria as follows: >10 AFB /field ------+++ 1-10 AFB /field ---------++ 10-100 AFB /100 field ---+ 1-9 AFB /100 fields ---- report the exact number.

Morphology of the bacteria (cocci, diplococci, streptococci, rods or coccobacilli). Presence and no. of pus cells Presence of yeast cells and epithelial cells.

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When no AFB is seen, report the smear as No AFB seen.

Endospores Staining (Schaeffer-Fulton Method) Objective: To stain bacterium to observe the endospores. Principle: Spores have a characteristic appearance under the microscope and their development follows a regular pattern. Spore development can be followed in the microscope by doing simple stains (spores appear colorless) or special spore stains (spores appear colored). Endospores have a thick wall, which makes them quite resistant to penetration by harmful chemicals. That same thick wall also makes the spores resistant to staining. A single unstained area within a cell almost as wide as the cell itself is likely to be a spore. Endospores can be positioned terminally, sub-terminally, or centrally within the cell. Materials 3- to 5-day-old agar slant culture of Bacillus subtilis 24-hour-old slant culture of Staphylococcus epidermidis Malachite green solution Safranin solution Slides Diamond glass-marking pencil Slide rack 500-ml beaker Tripod with asbestos mat Forceps Procedures 1. Place a drop of water on a slide. Emulsify a small amount of each of the slant cultures in the same drop of water. 2. Ring and code the slide with the marking pencil. 3. Stain the slide by the endospore stain: 4. Place the slides on a slide rack extended across a beaker of boiling water held on a tripod (an electric burner may be used instead of a Bunsen burner). An asbestos mat should protect the beaker from the Bunsen flame beneath. 5. Flood slide with malachite green and allow to steam gently for 5 to 10 minutes. The stain itself should not boil; if it does, reduce the heat. If the stain appears to be evaporating and drying too rapidly, add a little more. Keep the slide flooded. 6. Allow the slide to cool slightly, then use forceps to drain the slide over a sink or staining tray and rinse with water for about 30 seconds until no more green washes out. 7. Counterstain the preparation with safranin for 30 seconds, then rinse again with tap water. Blot or air-dry the slide. 8. Examine the smear under the oil-immersion objective and record your observations under Results 1. In the circle, make a sketch of your microscopic observations. Note the difference between the staining properties of the staphylococci and the endospores. Use colored pencils if they are available. Indicate the color of the spores in contrast to the vegetative cells. Flagella Staining Objective: To stain bacterium for observation of flagella Principle: The small diameter of the flagellum makes it impossible to see in the light microscope without the addition of a large amount of dye. Because there is still no easy or reliable method for staining flagella, you will view prepared slides of bacteria with flagella. However, the effect of the rotation of the flagellum can be observed by tethering a cell to the surface of a slide coverslip by its flagellum. With the distal end of the flagellum fixed on the surface of the coverslip the rotation of the hook causes the cell to rotate in place. This cell rotation can be observed in the microscope. Materials: One overnight culture of K. pneumoniae in milk One overnight culture of E. coli in NB 20% copper sulfate solution Procedure: 1. Obtain a culture of K. pneumoniae grown in milk and aseptically remove a loopful of the culture. 2. Spread the inoculum on a slide, making a thin smear over one half of the slide. 3. Obtain a loopful of E. coli and make a thin smear on the other half of the slide. 4. Let the slide air dry. 5. Flood the smear with aqueous crystal violet and let stand 2 minutes. 6. Wash the slide with copious quantities of 20% copper sulfate solution and blot dry.

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7. 8.

Examine the smear under oil immersion, searching for an area that is thinly stained. Draw and label the cells and their surrounding flahella,

Capsule staining Objective: To stain bacterium for observation of capsules Principle: All bacteria probably have a certain amount of capsular material surrounding the cell. In many cases, however, the layer is not large enough to be readily discernible whereas some species elaborate copious slime layers readily visible in the microscope. Their composition varies according to species, some consist of polysaccharides, others appear to be made of glycoprotein, and some are polypeptides. Capsular subunits are synthesized between the cell membrane and the cell wall and are inserted into the preexisting capsule outside the cell wall. Materials: One overnight culture of K. pneumoniae in milk One overnight culture of E. coli in NB 20% copper sulfate solution Procedure: 1. Obtain a culture of K. pneumoniae grown in milk and aseptically remove a loopful of the culture. 2. Spread the inoculum on a slide, making a thin smear over one half of the slide. 3. Obtain a loopful of E. coli and make a thin smear on the other half of the slide. 4. Let the slide air dry. 5. Flood the smear with aqueous crystal violet and let stand 2 minutes. 6. Wash the slide with copious quantities of 20% copper sulfate solution and blot dry. 7. Examine the smear under oil immersion, searching for an area that is thinly stained. 8. Draw and label the cells and their surrounding capsules, include this in your question section. Results: 7. DISINFECTION AND STERILIZATION There are a number of agents available for the control of microbial growth. Some of these agents cause physical changes in the environment. Physical agents include X-rays, ultraviolet irradiation, temperature and high solute concentrations that cause changes in osmotic pressure. Chemical agents, include acids, bases, alcohols, phenols, halogens, heavy metals, and quartenary ammonium compounds (a special class of detergents). Chemotherapeutic agents, which are used to treat microbial infections in humans and animals, are a special class of chemical agents suitable for use within a eukaryotic host. Control agents and the control process are described by a number of terms such as sterilization, disinfection, and so on. A number of these terms are defined below: Sterilization: A treatment that makes an object completely free of all viable microorganisms. Disinfection: A chemical or physical treatment that reduces the infectivity of an object by killing most of the harmful cells present, but not necessarily the spores present. Antimicrobial agent: A chemical or physical treatment that interferes with the growth or activity of microorganisms. Chemotherapeutic agent: An antimicrobial agent used for treating infections. Antibiotics are chemotherapeutic agents. Sanitization: Disinfection designed to meet public health requirements. Thermal death point: The lowest temperature at which a 10 minute exposure sterilizes a culture. Methods A. Physical Method B. Chemical Method Physical Method Moist Heat Sterilization Objective: To demonstrate destruction of microorganisms by moist heat applied under controlled conditions of time and temperature Principle: It is possible to quantitate the response of microorganisms to heat by measuring the time required to kill them at different temperatures. The lowest temperature required to sterilize a standardized pure culture of bacteria within a given time (usually 10 minutes) can be called the thermal death point of that species, and, conversely, the time required to sterilize the culture at a stated temperature can be established as the thermal death time. Materials: Tubed nutrient broths (5-ml aliquots) Nutrient agar plates Sterile 1.0-ml pipettes

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24-hour broth culture of Staphylococcus epidermidis Six-day-old broth culture of Bacillus subtilis Procedures 1. Set up a beaker water bath and heat to boiling. 2. Divide one nutrient agar plate in half by marking the bottom of the plate with a wax pencil or ink marker. 3. Streak a loopful of the S. epidermidis culture onto one-half of the nutrient agar plate. Label the section of the plate with the name of the organism and the word Control. 4. Repeat step 3 with the culture of B. subtilis, inoculating the second half of the plate. 5. Place the control plate in the 35C incubator for 24 hours. 6. Divide two nutrient agar plates into 4 quadrants by marking the bottom of the plates with a wax pencil or ink marker. Label one plate S. epidermidis and the other B. subtilis. Label the 4 quadrants on each plate as follows: 5, 10, 15, 30 minutes. 7. Take a pair of broth tubes and inoculate each, respectively, with 0.1 ml of S. epidermidis and B. subtilis. Place these tubes in the boiling water bath. Note the time. 8. Leave the pair of broth cultures in boiling water for 5 minutes. Remove the tubes and cool them quickly under running cold tap water. Streak a loopful of each boiled culture onto the quadrant of nutrient agar labeled 5 minutes. 9. Return the tubes to the boiling water bath for an additional 5 minutes. Begin timing when the water comes to a full boil. Cool the tubes as in step 8 then streak a loopful of each culture onto the quadrant of nutrient agar labeled 10 minutes. 10. Repeat step 9 twice more, streaking loopfuls of culture onto the quadrants of the plates labeled 15 and 30 minutes, respectively. 11. Incubate subcultures from boiled tubes at 35C for 24 hours. Results 1. Read all plates for growth or no growth. Record your results in the chart. Dry heat sterilization Purpose: To compare the effects of moist and dry heat Principle: In this experiment, egg white (the protein, albumin) is used to simulate microbial enzyme protein. The speed of the damaging reaction (coagulation) of moist and dry heat on protein will be observed. Materials Tubed distilled water (0.5-ml aliquots) Sterile 1.0-ml pipettes Clean tubes Dry-heat oven Egg white (albumin, a protein) Procedures 1. Set up a beaker water bath and heat to boiling. 2. Set the dry-heat oven for 100C. 3. Using a pipette, measure 0.5 ml of egg white into 0.5 ml of distilled water. 4. Place the tube into the boiling water bath and immediately begin timing. Observe until the egg white has coagulated, then record the elapsed time. 5. Using a pipette, measure 1.0 ml of egg white into a clean tube. 6. Place the tube into the dry-heat oven and immediately begin timing. Observe until the egg white has coagulated, then record the elapsed time. Results 1. Elapsed time for protein coagulation in moist heat (boiling): Elapsed time for protein coagulation in dry heat (baking): 2. State your interpretation of the effect of moisture on protein denaturation

Ultraviolet Irradiation Objective: To observe the effect of UV-radiation for different periods on bacterial cells

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Principle: Ultraviolet light (175 to 350 nanometers in wavelength) has a higher energy than visible light. Ultraviolet light (UV) of about 260 nanometers wavelength is strongly absorbed by the DNA molecules in all cells. DNA may be structurally changed as a result. If the DNA suffers multiple UV hits, there are several possible outcomes: (1) cell division may stop while the cell repairs the UV damage to its DNA (a bacteriostatic effect); (2) the cell may lose viability (a bactericidal effect) because it is unable to repair its DNA which can no longer function in replication (DNA synthesis) or expression (mRNA synthesis); (3) the cell may make a mistake during the repair of UV damage resulting in a viable cell carrying a mutation in a gene. There are two limitations to the use of UV as a control agent which must be kept in mind: (1) UV penetrates poorly. It is only effective on surfaces or thin layers of liquid. To irradiate plates you must remove the lids. (2) UV can damage your eyes. Never look directly at an ultraviolet lamp. Materials: Dilution blanks of 9.0 or 9.9 ml water 1 overnight culture of Serratia marcescens UV light source 6 NA plates Semi-log graph paper Procedure 1. Dilute the culture so that you can inoculate 100-300 bacterial cells per plate. Spread inoculum evenly over the surface of each of the 6 plates. (Do not use more than 0.2 ml inoculum). 2. At a distance of 18 inches from the UV light source, irradiate 2 plates each of the times listed below. Make sure lids are removed. 3. Label each plate. Place one plate from each pair in a paper bag immediately (or wrap in paper) and place in the 30 o C incubator. 4. Place the other plate from each pair under a regular lamp for 10-15 minutes. Make sure lids are on. 5. Place these plates also in the incubator. 6. Examine after 48 hours and record the number of colonies per plate in your notes. Make a table like the one shown. 7. Plot curves of number of survivors per milliliter as a function of UV dose with and without photoreactivation (regular light) on the same sheet of graph paper. Time of UV exposure 0 seconds 10 seconds 20 seconds Chemical Methods A wide variety of chemical agents display antimicrobial activity to some degree. In considering their application to patient care, we may separate them into two general classes: (1) those that are useful for destroying pathogenic microorganisms in the environment (disinfectants) or on skin (antiseptics), and (2) those that may be administered to patients for treatment of infectious diseases (antimicrobial agents). Many antimicrobial substances are too toxic to be used for patient therapy but are valuable as environmental disinfectants. Each agent has a limited chemical mode of action, and microorganisms exposed to it may vary widely in their responses. Some microbes or their forms may succumb to its effects (such as vegetative bacterial cells) whereas others may not (such as bacterial endospores). Antimicrobial Activity of Disinfectants Objective: To observe the effect of antimicrobial activity disinfectants Materials: Overnight cultures of Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Streptococcus faecalis 3 NA plates Various disinfectant and antiseptic solutions Sterile filter discs Procedure 1. Spread out 0.1 ml of the overnight culture of one of the above culture on all 3 NA plates. 2. Using sterile/flamed forceps obtain a sterile filter disc and immerse it in one of the disinfectants or antiseptics (beaker provided). 3. Place the disc on one of the plates that you just spread with an organism. Number of colonies No photoreactivation Number of colonies + photoreactivation

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4.

Repeat steps 2 and 3 until you have 3 discs with different solution on them in each of your 3 plates (a total of nine discs on three plates). Label the plates with organism and type of solution on each disc. 5. Incubate all 3 plates at 30o C for 48 hours. 6. Observe the plates and determine if the solution inhibited growth of the bacteria. Measure the zone of inhibition (if present). Create a table with organism used, type of disinfectant, and zone of inhibition. Include this in your answers to the questions. Checking for disinfection power of ethyl alcohol Objective: To cheek disinfection power of ethyl alcohol at different concentration. Materials required: - ethyl alcohol (25%, 50%, 70% and 96%) - known bacteria suspension. - Pasture pipette - Culture media - Wire loop - Sprit lamp Procedure: - Prepare ethyl alcohol at concentrations of 25%, 50%, 70% and 96%. -Prepare uniform suspension of known bacterial colony. -Introduce equal volume of bacterial suspension (2 drops) into 1ml of each concentration of Ethyl alcohol - Wait for 2 min. -Subculture into a sterile culture medium separately for each concentration. - Incubate for 24 hours at 37oc. Result: Look for the difference in density of bacterial colony. 8. MEDIA AND CULTURING OF MICRO-ORGANISMS Preparation of culture media Objective: To prepare the culture media Materials required: Dehydrated nutrient agar Dehydrated nutrient broth A balance, and weighing papers A 1-liter Erlenmeyer flask, cotton plugged or screw capped A 1-liter glass beaker A 1-liter graduated cylinder Glass stirring rods (at least 10 cm long) 10-ml pipettes (cotton plugged) Test tubes (screw capped or cotton plugged) Petri dishes Aspiration device for pipetting Procedures 1. Read the label on a bottle of dehydrated nutrient agar. It specifies the amount of dehydrated powder required to make 1 liter (1,000 ml) of medium. Calculate the amount needed for 1/2 liter and weigh out this quantity. 2. Place 500 ml of distilled water in an Erlenmeyer flask. Add the weighed, dehydrated agar while stirring with a glass rod to prevent lumping. 3. Set the flask on a tripod over an asbestos mat. Using a Bunsen flame, slowly bring the rehydrated agar to a boil. Stir often. An electric hot plate may be used instead of a Bunsen burner. 4. When the agar mixture is completely dissolved, remove the flask from the flame or hot plate, close it with the cotton plug or cap, and it has to be sterilized in the autoclave. 5. While the flask of agar is being sterilized, prepare 500 ml of nutrient broth, adding the weighed dehydrated powder to the water in a beaker for reconstitution and dissolution. 6. Bring the reconstituted broth to a boil, slowly. When fully dissolved, remove from flame or electric burner and allow cooling a bit. 7. Using a pipette, dispense 5-ml aliquots of the broth into test tubes (plugged or capped). Then sterilize them.

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8.

When the flask of sterilized agar is returned to you, allow it to cool to about 50C (the agar should be warm and melted, but not too hot to handle in its flask). Remove the plug or cap with the little finger of your right hand and continue to hold it until you are sure it wont have to be returned to the flask. Quickly pour the melted, sterile agar into a series of petri dishes. The petri dish tops are lifted with the left hand, and the bottoms are filled to about one-third capacity with 9. melted agar . Replace each petri dish top as the plate is poured. When the plates are cool (agar solidified), invert them to prevent condensing moisture from accumulating on the agar surfaces. 10. Place inverted agar plates and tubes of sterilized nutrient broth in the 35C incubator. They should be incubated for at least 24 hours to ensure they are sterile (free of contaminating bacteria) before you use. Results: Inoculation of culture media The artificial induction of microorganisms into a medium is called inoculation. The latter is the most fundamental technique for studying the growth characteristics of micro-organisms and for transfer and maintenance of culture under aseptic conditions. Following three are the most commonly used methods of pure cultures in routine laboratory work: (i) Streak plate method (ii) Pour plate method (iii) Spread plate method. Streak plate method Objective: To prepare streak plates for obtaining pure culture Principle: The colonies on mixed plate are separated by spreading on a plate with good spacing among each other using streak plate method. Materials Nutrient agar plates Blood agar plates Sterile swabs A mixed broth culture A demonstration plate culture made from this broth, showing colonies isolated by good Streaking technique Glass slides

Procedures: A. Streaking a Mixed Broth Culture for Colony Isolation 1. Make certain the contents of the broth culture tube are evenly mixed. 2. Place a loopful of broth culture on the surface of a nutrient agar plate, near but not touching the edge. With the loop flat against the agar surface, lightly streak the inoculum back and forth over approximately one-eighth the area of the plate; do not dig up the agar 3. Sterilize the loop and let it cool in air. 4. Rotate the open plate in your left hand so that you can streak a series of four lines back and forth, each passing through the inoculum and extending across one side of the plate 5. Sterilize the loop again and let it cool in air. 6. Rotate the plate and streak another series of four lines, each crossing the end of the last four streaks and extending across the adjacent side of the plate 7. Rotate the plate and repeat this parallel streaking once more 8. Finally, make a few streaks in the untouched center of the plate. Do not touch the original inoculum. 9. Incubate the plate (inverted) at 35C. B. Throat/oral swab culture 1. Rotate a sterile swab over the surface of your tongue and gums. 2. Roll the swab over a small -cm square of surface of a blood agar plate, near but not touching one edge 3. Rotate the swab fully in this area. Discard the swab in a container of disinfectant. 4. Using an inoculating loop, streak the plate 5. Incubate the plate (inverted) at 35C. Results:

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Pour plate method Objective: To prepare pour plate to obtain pure culture Principle: An alternative method for using agar plates to obtain isolated colonies, other than streaking their surfaces, is to prepare a pour plate. In this case, an aliquot of the specimen to be cultured is placed in the bottom of an empty, sterile petri dish, then melted, cooled agar is poured over it. Quickly, before the agar cools, the plate is gently rocked to disperse the inoculum. When the agar has solidified and the plate is incubated, any bacteria present in the specimen will grow wherever they have been embedded within the agar layer or localized on its surface. Their colonies will be isolated and can be removed from subsurface positions by inserting the inoculating loop or a straight wire into the agar. To prepare pour plates, the inoculum must be a liquid specimen or culture. If it is not, it must be suspended in sterile fluid before being placed in the petri dish. Materials Tubed nutrient agar (10 ml per tube) Sterile petri dishes Sterile 1-ml pipettes (cotton plugged) Mixed broth culture containing Escherichia coli and Staphylococcus epidermidis Nutrient agar plates Nutrient agar broth Procedures 1. Place a tube of sterile nutrient agar in a boiling water bath. 2. When the agar is liquefied, remove the tube and allow it to cool to about 50C. 3. Place an empty sterile petri dish before you, top side up. 4. Remove a sterile 1-ml pipette from its container, keeping your fingers on the plugged mouth end. 5. Pick up the mixed broth culture in the other hand, remove its closure with the little finger of the hand holding the pipette (do not touch the pipette to anything), and insert the pipette into the broth. 6. Holding the tube and pipette vertically, poise your index finger over the pipette mouth. Allow the pipette to fill to the level of the broth in the tube and then close off its mouth with your finger (there should be about 0.3 to 0.4 ml of culture in the pipette). 7. Keeping your finger pressed on its top, raise the pipette until the tip is free of the broth and then slowly allows the material in the pipette to run back into the tube until only the last 0.1 ml remains. Now press your finger tightly to close the pipettes mouth and prevent further dripping. Never use your mouth to draw fluid into a pipette. 8. Before you withdraw the pipette from the tube, touch its tip against the dry inner wall to remove any drop hanging from it. 9. Withdraw the closed pipette, replace the tube closure, and put the tube down in the rack. 10. Now, with your free hand, remove the top of the petri dish (do not put it down), place the tip of the pipette against the bottom of the dish, release your finger from the mouth, and let 0.1 ml of broth culture run into the plate bottom. 11. Replace the dish top and discard the pipette into a container of disinfectant. 12. Pick up the tube of melted, cooled agar, remove its closure, and put it down on the bench top. 13. With your free hand, remove the top of the petri dish (again, do not put it down). Quickly pour the agar into the dish. 14. Replace the petri dish cover (the tube may be set aside for washing). Gently rock the closed dish, or rotate it in circular fashion on the bench top, being careful not to allow the still melted agar to wave up over the edge of the bottom half or onto the cover. 15. Let the agar solidify without further disturbance. When it is quite firm (about 30 minutes), invert the plate and place it in the 35C incubator.

Results: Spread plate method. Objective: To prepare spread plate to obtain pure culture The spread plate technique is employed for isolation of pure culture from mixed colonies. The propagules of microorganisms are spread over solidified agar medium with the help of bent glass rod called spreader. Materials Sterile petri dishes Sterile 1-ml pipettes (cotton plugged) Mixed broth culture containing Escherichia coli and Staphylococcus epidermidis Nutrient agar plates

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Nutrient agar broth Bent gloss rod 1. 2. Place a sterile agar petri dish before you, top side up. Remove a sterile 1-ml pipette from its container, keeping your fingers on the plugged mouth end. 3. Pick up the mixed broth culture in the other hand, remove its closure with the little finger of the hand holding the pipette (do not touch the pipette to anything), and insert the pipette into the broth. 4. Holding the tube and pipette vertically, poise your index finger over the pipette mouth. Allow the pipette to fill to the level of the broth in the tube and then close off its mouth with your finger (there should be about 0.3 to 0.4 ml of culture in the pipette). 5. Keeping your finger pressed on its top, raise the pipette until the tip is free of the broth and then slowly allow the material in the pipette to run back into the tube until only the last 0.1 ml remains. Now press your finger tightly to close the pipettes mouth and prevent further dripping. Never use your mouth to draw fluid into a pipette. 6. Before you withdraw the pipette from the tube, touch its tip against the dry inner wall to remove any drop hanging from it. 7. Withdraw the closed pipette, replace the tube closure, and put the tube down in the rack. 8. Now, with your free hand, remove the top of the petri dish (do not put it down), place the tip of the pipette against surface of the dish, release your finger from the mouth, and let 0.1 ml of broth culture run into the plate bottom. 9. Replace the dish top and discard the pipette into a container of disinfectant. 10. Pick up the bent gloss rod, sterilize by exposing to burning flame or alcohol, allow it to cool. 11. With your free hand, remove the top of the petri dish (again, do not put it down). Slowly rotate the gloss rod to spread the inoculums along the surface of the petridish. 12. Close the lid and keep in incubator at 37C in an inverted position. Results: Culturing microorganisms from hospital environment Purpose To take cultures from selected areas of the hospital environment, in order to identify sources of contaminating microorganisms Principle: Microorganisms are found throughout the environment: in the air and water; on the surface of objects, clothes, tables, floors; in soil and dust; and on the skin and mucous membranes of our own bodies. These widely present microorganisms ordinarily are of no concern to healthy humans, provided we maintain good hygiene in our daily living. In hospitals, however, where susceptible patients must be protected from hospital-acquired (nosocomial ) infections, the concentration and distribution of microorganisms in the environment are of great importance. Frequent monitoring of the environment is one of the responsibilities of the hospital epidemiologist, who may be a microbiologist, nurse, or physician. Materials Nutrient broth Nutrient agar plates Sterile swabs Procedures 1. Place a swab in a nutrient broth to moisten it. As you withdraw the swab, press it against the inner wall of the tube to drain off excess fluid. 2. Take a culture of the floor with this swab by rubbing and rotating it over an area approximately 10 cm square. 3. Inoculate an agar plate with the swab by rotating it over a small area near one edge. Discard the swab and use your wire loop to streak out the plate in a manner to obtain isolated colonies. 4. Moisten another swab in broth and take a culture of the sink faucet in the area around the aerator or strainer. Inoculate and streak another agar plate as in step 3. 5. Take a fresh agar plate and touch separate areas of the agar surface with each fingertip of your right hand. 6. Take an agar plate into the lavatory. Place it on a shelf or the basin, remove the top, and leave the agar exposed for 30 minutes. Close, invert, and incubate the plate at 35C. 7. Look around the laboratory for any area where dust has accumulated (window ledges, open shelves, hard-to-clean areas). Take a culture of dust with a moist swab, inoculate, and streak an agar plate. 8. Take a culture (with a moist swab) of a 5-cm square area on the front of your laboratory coat. Inoculate and streak a plate. 9. Run a moist swab through your hair. Inoculate and streak a plate. 10. Incubate all plates, inverted, at 35C. Results:

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Examine all plates and record your observations in the table.

8. BIOCHEMICAL TESTING In most situations where identification is necessary, the bacterium is not completely unknown; instead, evidence for the presence of a bacterium from a limited group of possibilities is sought as in the following examples: 1. A medical laboratory wishes to confirm the presence or absence of a bacterium from a particular pathogenic microorganism to help in diagnosis. 2. A food laboratory looks for particular microorganisms known to be associated with specific food products. 3. A government laboratory wants to determine the presence or absence of particular microorganisms to measure the effectiveness of sewage treatment or the degree of pollution of a water source. 4. A research laboratory is interested in isolating an organism that performs a specific task, such as photosynthesis or the degradation of a pesticide. The easiest way to obtain a pure culture is by use of a streak plate. The cells must be spread out on a plate so that on some portion of it they are separated from each other and can form isolated colonies. An isolated colony is then restreaked to check its purity, and a colony from this second streak is transferred to a slant for use as the master culture for the identification. Simple streaking on complex medium, however, may not be sufficient for obtaining a pure culture from nature because (1) many different microorganisms may be present, and (2) the microorganism may be present in a small proportion relative to other microorganisms. Instead, either a selective enrichment or a differential medium is used as a first step before streaking on complex medium. Carbohydrate Metabolism test Principle: Different bacteria can ferment a wide variety of sugars and other compounds. Several types of acids, alcohols, and gases may be produced. These products are used as indicators of fermentation. The determination of a fermentation pattern with a series of different carbon sources, usually sugars, by an unknown bacterial species is a valuable aid in identifying that species. The production of acid or alkaline products causes a noticeable color change in the pH indicator, which is included in the medium. Gas production is determined by the inclusion of a small, inverted Durham tube in the culture tube. When gas is produced, it is trapped in the Durham tube and can be seen as a bubble. Materials: Overnight culture of each of the following: S. aureus, S. epidermidis, E. coli, E. aerogenes, S. marcescens, P. vulgaris tubes of the following media (broth or slant) Mannitol fermentation broth Sucrose fermentation broth Lactose fermentation broth Figure: Carbohydrate Fermentation (a) Possible carbohydrate fermentation patterns of microorganisms, with phenol red as the pH indicator. (b) The tube on the left is the control. The next tube shows alcohol fermentation. Notice the gas bubble at the top. The third tube from the left shows no carbohydrate fermentation (negative). The tube on the right shows acid and gas production Procedure: 1. Inoculate each of the broth media with a loopful of the organisms separately. 2. Inoculate the agars with a stab then a streak. 3. Incubate for 48 hours at 37o C. 4. Observe growth and/or color change in the media. 5. Determine unknown bacteria by comparing to chart. Result: Citric Acid Utilization test Principle: Citric acid is an intermediate in the tricarboxylic acid (TCA) cycle that oxidizes pyruvate to carbon dioxide in respiration. To use citric acid, a bacterium must be able to transport it across the cell membrane. Utilization of citrate (the salt of citric acid) differentiates between some enteric bacteria. This test is performed to determine whether a bacterium can use citric acid as its sole carbon source. It is a practical test for distinguishing between E. coli, a fecal organism which cannot use citrate as the sole carbon source, and Enterobacter aerogenes, a soil organism often found in

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water that can. Since E. coli is an indication of fecal contamination and E. aerogenes is not, this is a routine test for examining water quality. The test is also useful in distinguishing between various pathogenic Salmonella species. Materials: 1 overnight culture of each of the following: S. aureus, S. epidermidis E. coli, E. aerogenes, S. marcescens, P. vulgaris tubes of the following media (broth or slant) Simmons citrate agar Procedure: 1. Inoculate each of the broth media with a loopful of the organisms separately. 2. Inoculate the agars with a stab then a streak. 3. Incubate for 48 hours at 37o C. 4. Observe growth and/or color change in the media. 5. Determine unknown bacteria by comparing to chart. Urease Test Principle: Urea is a nitrogenous waste product of animals. Some bacteria can cleave it to produce carbon dioxide and ammonia. The production of ammonia raises the pH of the media. The indicator phenol red is utilized in the broth, which is orange-yellow at pH <6.8, and turns pinkish-red at pH >8.1. Hence a positive urea test is denoted by the change of media color from yellow to red. Urease test distinguishes between Proteus vulgaris (positive) and E. coli and S. marcescens (negative). Materials: 1 overnight culture of each of the following: S. aureus, S. epidermidis E. coli, E. aerogenes, S. marcescens, P. vulgaris, tubes of the Urease broth Procedure: 1. Inoculate each of the broth media with a loopful of the organisms separately. 2. Inoculate the agars with a stab then a streak. 3. Incubate for 48 hours at 37o C. 4. Observe growth and/or color change in the media. 5. Determine unknown bacteria by comparing to chart.

Catalase Test Principle: Catalase is an enzyme that decomposes hydrogen peroxide into oxygen and water. Excluding the Streptococci, most aerobic and facultative anaerobic bacteria possess catalytic activity. Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism. Catalase converts hydrogen peroxide into water and oxygen. The catalase test is commonly used to differentiate streptococci (negative) for staphylococci (positive). Materials Well -isolated colonies of an 18-24 hour culture. 3% Hydrogen peroxide, Clean glass slide, Dropper, Bacteriological Loop Controls Positive: Staphylococcus aureus Negative: Streptococcus sp. Procedure: 1. With loop or applicator stick, transfer cells from the center of a well-isolated colony to a glass slide. 2. Add 1-2 drops of the 3% Hydrogen peroxide to the bacterial cells Note: It is recommended to use this order instead particularly if iron containing loops are used, due to false positives Interpretation Positive: rapid, appearance of sustained gas bubbles Negative: No gas bubble production of adding the organism to the reagent,

Coagulase test Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase are produced by most strains of S. aureus: _ Free coagulase which converts fibrinogen to fibrin by activating a coagulase-reacting factor present in plasma. Free coagulase is detected by clotting in the tube test. Bound coagulase (clumping factor) which converts fibrinogen directly to fibrin without requiring a coagulasereacting factor. It can be detected by the clumping of bacterial cells in the rapid slide test. A tube test must always be performed when the result of a slide test is not clear, or when the slide test is negative and Staphylococcus has been isolated from a

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serious infection. A tube test may be required to detect some MRSA (methicillin resistant S. aureus) strains although some commercially available latex test kits to differentiate coagulase positive and coagulase negative staphylococci, overcome this. Before performing a coagulase test, examine a Gram stained smear to confirm that the organism is a Gram positive coccus. Materials: EDTA anticoagulated human plasma or rabbit plasma. The plasma should be allowed to warm to room temperature before being used Plasma: Oxalate or heparin plasma can also be used. Do not use citrated plasma because citrate-utilizing bacteria e.g. enterococci, Pseudomonas and Serratia may cause clotting of the plasma (in tube test). Occasionally, human plasma may contain inhibitory substances which can interfere with coagulase testing. It is therefore essential to test the plasma using a known coagulase positive S. aureus. The plasma can be stored frozen in amounts ready for use. Slide test method (detects bound coagulase) Procedure: 1 Place a drop of distilled water on each end of a slide or on two separate slides. 2 Emulsify a colony of the test organism (previously checked by Gram staining) in each of the drops to make two thick suspensions. Note: Colonies from a mannitol salt agar culture are not suitable for coagulase testing. The organism must first be cultured on nutrient agar or blood agar. 3 Add a loopful (not more) of plasma to one of the suspensions, and mix gently. Look for clumping of the organisms within 10 seconds. No plasma is added to the second suspension. This is used to differentiate any granular appearance of the organism from true coagulase clumping. Results Clumping within 10 secs . . . . . . .. . . . . . S. aureus No clumping within 10 secs . . . .No bound coagulase Note: Virulent strains of Yersinia pestis are also coagulase positive. Controls Positive coagulase control: Staphylococcus aureus Negative coagulase control: Escherichia coli or Staphylococcus epidermidis Tube test method (detects free coagulase) Procedure: 1. Take three small test tubes and label: T _ Test organism (1824 h broth culture) Pos _ Positive control (1824 h S. aureus broth culture) Neg _ Negative control (sterile broth) 2 Pipette 0.2 ml of plasma into each tube. 3 Add 0.8 ml of the test broth culture to tube T. Add 0.8 ml of the S. aureus culture to the tube labelled Pos. Add 0.8 ml of sterile broth to the tube labeled Neg. 4 After mixing gently, incubate the three tubes at 3537 _C. Examine for clotting after 1 hour. If no clotting has occurred, examine after 3 hours. If the test is still negative, leave the tube at room temperature overnight and examine again. Note: When looking for clotting, tilt each tube gently. Results Clotting of tube contents or . .. . . . . . . . S. aureus fibrin clot in tube No clotting or fibrin clot . . . . . . . . Negative test Note: There should be no clotting in the negative control tube. Commercially produced agglutination tests to identify S. aureus Several latex agglutination test kits have been developed to identify S. aureus based on the detection of clumping factor, and, or protein A.

Bile solubility test Principle

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A heavy inoculum of the test organism is emulsified in physiological saline and the bile salt sodium deoxycholate is added. This dissolves S. pneumoniae as shown by a clearing of the turbidity within 1015 minutes. Viridans and other streptococci are not dissolved and therefore there is no clearing of the turbidity. This helps to differentiate S. pneumoniae, which is soluble in bile and bile salts, from other alpha-haemolytic streptococci (viridans streptococci) which are insoluble. Materials: Sodium deoxycholate, 100 g/l (10% w/v) Physiological saline (sodium chloride, 8.5 g/l) Tube method Although the bile solubility test can be performed by testing colonies directly on a culture plate or on a slide (see subunit 7.18.4), a tube technique is recommended because the results are easier to read. 1 Emulsify several colonies of the test organism in a tube containing 2 ml sterile physiological saline, to give a turbid suspension. 2 Divide the organism suspension between two tubes. 3 To one tube add 2 drops of the sodium deoxycholate reagent and mix. 4 To the other tube (negative control), add 2 drops of sterile distilled water and mix. 5 Leave both tubes for 1015 minutes at 35 37 _C. 6 Look for a clearing of turbidity in the tube containing the sodium deoxycholate Results Clearing of turbidity . . . . . . . . . . . . . . . . . Probably S. pneumoniae No clearing of turbidity . . . . Organism is probably not S. pneumoniae There should be no clearing of turbidity in the negative control tube to which distilled water was added. Note: Some strains of S. pneumoniae are not dissolved by bile salts, and very occasionally some strains of viridans streptococci give a positive test. Controls Bile solubility positive control: Streptococcus pneumoniae Bile solubility negative control: Enterococcus Faecalis 8.8 DNA-ase test Principle: Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA). The test organism is cultured on a medium which contains DNA. After overnight incubation, the colonies are tested for DNA-ase production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates unhydrolyzed DNA. DNA-ase-producing colonies are therefore surrounded by clear areas due to DNA hydrolysis. This test is used to help in the identification of S. aureus which produces deoxyribonuclease (DNAase) enzymes. The DNA-ase test is particularly useful when plasma is not available to perform a coagulase test or when the results of a coagulase test are difficult to interpret. Materials DNA-ase agar plate, Up to six organisms may be tested on the same plate. Hydrochloric acid 1 mol/1 (1 N) Procedure: 1 Divide a DNA-ase plate into the required number of strips by marking the underside of the plate. 2 Using a sterile loop or swab spot-inoculate the test and control organisms. Make sure each test area is labelled clearly. 3 Incubate the plate at 3537 _C overnight. 4 Cover the surface of the plate with 1 mol/l hydrochloric acid solution. Tip off the excess acid. 5 Look for clearing around the colonies within 5 minutes of adding the acid, Results Clearing around the colonies . . . . . . . . DNA-ase positive strain No clearing around the colonies . . . . . DNA-ase negative strain Note: Some methicillin resistant S. aureus (MRSA) strains give a negative DNA-ase test. Some coagulase negative staphylococci are weakly positive. Also, S. pyogenes, Moraxella and Serratia species frequently give a positive DNA-ase test. Controls Positive DNA-ase control: Staphylococcus aureus Negative DNA-ase control: Staphylococcus Epidermidis

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Aesculin hydrolysis test to identify enterococci Principle: This test can be economically performed using a bile aesculin tablet . Procedure: The test can be performed by placing a tablet on a blood agar plate inoculated with the test organism and incubating it at 3537 C overnight. A positive test is indicated by the tablet and colonies around it turning black/grey. A negative test is shown by the tablet remaining white and no change in colour of the colonies. A zone of inhibition may appear around the tablet. Alternatively, the test can be performed by making a dense suspension of the test organism in 0.25 ml of physiological saline in a small tube, adding a tablet, and incubating at 3537 C for 4 hours (or overnight). A positive reaction is shown by a black/grey colour in the medium. Note: An aesculin hydrolysis can also be performed by incubating the test organism on bile aesculin agar but this medium is expensive.

Oxidase test Principle A piece of filter paper is soaked with a few drops of oxidase reagent. A colony of the test organism is then smeared on the filter paper. Alternatively an oxidase reagent strip can be used . When the organism is oxidase-producing, the phenylenediamine in the reagent will be oxidized to a deep purple colour. Occasionally the test is performed by flooding the culture plate with oxidase reagent but this technique is not recommended for routine use because the reagent rapidly kills bacteria. It can however be useful when attempting to isolate N. gonorrhoeae colonies from mixed cultures in the absence of a selective medium. The oxidase positive colonies must be removed and subcultured within 30 seconds of flooding the plate. The oxidase test is used to assist in the identification of Pseudomonas, Neisseria, Vibrio, Brucella, and Pasteurella species, all of which produce the enzyme cytochrome oxidase. Materials Oxidase reagents freshly prepared or use an oxidase reagent strip) Note: Fresh oxidase reagent is easily oxidized. When oxidized it appears blue and must not be used. Stable oxidase reagent strips The strips have a 5 year shelf-life when stored at 28 C. Procedure (fresh reagent) 1 Place a piece of filters paper in a clean petri dish and add 2 or 3 drops of freshly prepared oxidase reagent. 2 Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test organism and smear it on the filter paper. 3 Look for the development of a blue-purple colour within a few seconds Results Blue-purple colour . . . . . . . Positive oxidase test (within 10 seconds) No blue-purple colour . . . Negative oxidase test (within 10 seconds) Note: Ignore any blue-purple colour that develops after 10 seconds. Method using an oxidase reagent strip 1 Moisten the strip with a drop of sterile water. 2 Using a piece of stick or glass rod (not an oxidized wire loop) remove a colony of the test organism and rub it on the strip. 3 Look for a red-purple colour within 20 seconds. Red-purple colour . . . .. . . positive oxidase test. Controls Positive oxidase control: Pseudomonas aeruginosa Negative oxidase control: Escherichia coli Fibrinolysin Test Principle: The fibrinolysin test is used to determine the presence of a fibrinolytic enzyme which can dissolve fibrin clots. The fibrinolysin (a.k.a., staphylokinase) produced by most strains of Staphylococcus aureus, as well as, the streptokinases produced by virulent group A b-hemolytic Streptococcus (Streptococcus pyogenes) are examples of fibrinolytic enzymes,

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but are antigenically and enzymatically distinct from each other. The group C streptococci also produce an antigenically distinct fibrinolytic streptokinase and it is this particular enzyme that has been exploited commercially as the source of a thrombolytic (clot-busting) enzyme for clinical use in humans. Materials 24 hrs old S. epidermidis Rabbit plasma CaCl2 Procedure:

1. Prepare two test tubes, one without any bacterial inoculum and the other with S. epidermidis to demonstrate the
effects of a non-fibrinolysin producer. These tubes will be compared to the results obtained with the S. aureus strain, following prolonged incubation of the coagulase test, which will serve as an example of a positive fibrinolysin producer. In the first tube, CaCl2 (40 mmol/cc plasma) is added to ~0.5cc of platelet-rich plasma (~ >150 x 106 platelets/cc plasma) to produce a fibrin clot. The second tube is prepared identically, except that a generous loopful of the S. epidermidis strain is resuspended in the CaCl2-treated plasma. Thoroughly homogenize the inoculum with the loop and incubate the tube at 37o C for one to four hours. Examine the tube at 30 minute to hourly intervals for the first couple of hours for the presence of a clot by tipping the tube gently on its side. Reincubate the tube overnight to see if the clot subsequently lyses. In strains that produce fibrinolysin, the clot will be slowly digested.

2.

3.

Novobiocin susceptibility test Principle:The Novobiocin susceptibility test differentiates Staphylococcus, which is resistant to Novobiocin, from other coagulase negative Staphylococci (Example S. epidermidis), which are susceptible to Novobiocin. Materials: Sheep blood agar 5g Novobiocin disks Young culture of Staphylococcus epidermidis & Staphylococcus saprophyticus Procedure: 1. Streak the organism in three directions to form a lawn of growth on the sheep blood agar plate. 2. Place a 5g Novobiocin disc in the center of the inoculum 3. Incubate at 35 to 37 0c for 18 to 24 hours. 4. Examine the plate for inhibition around the disc. Measure the zone diameter in millimeters. Result: - Staphylococcus epidermidis Susceptible (Zone diameter greater than 16 mm) Staphylococcus saprophyticus Resistant (Zone diameter is less than or equal 16mm)

to

Bacitracin susceptibility test Principle: Group A -Hemolytic streptococci (Streptococcus pyogens) are susceptible to 0.04U Bacitracine while other members of the Streptococcus ( Example- S. pneumonia, S. agalactiae) are resistant to Bacitracine. Materials: - Blood agar plate - 0.04 Bacitracine disks Procedure: 1. Select a few of beta-hemolytic Streptococci to be tested and streak one-half the plate in three directions to obtain a lawn of growth. 2. Stab agar two or three times 3. Using sterile forceps, place on Bacitracine disk at the center of the streak. Tap gently with forceps. 4. Incubate 18-24 hours at 35 to 37 0C. Result: - Group A StreptococcusGroup Streptococcus and othersOptochin test Principle: In the presence of Optochin (Ethyl hydrocupreine hydrochloride), colonies of Streptococcus Pneumoniae are selectively lysed. Lysis is indicated by zone of inhibition after incubation under increased CO2. Other alpha-hemolytic Streptococci are resistant to Optochin and give a negative test. Positive (Bacitracine susceptible) Negative ( Bacitracine resistance)

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Materials: - Blood agar plates Optochin disks ( P Disks) Candle jar Streptococci pneumoniae Viridians streptococci Procedure: 1. Select 3 or 4 colonies of the 18 to 24 hour culture to be tested, and streak in three directions to obtain a lawn of growth. 2. Place an Optochin disk in the center of each streak. Gently tap with a sterile forceps. 3. Incubate in a candle jar for 24 hours at 35 to 37 0C. 4. Interpret plate for zones of inhibition. Streptococcus pneumoniae- Positive (Zone diameter greater than 14mm (10g disk) or greater than 10mm (6g disk)). Viridians Streptococci- Negative (Zone diameters less than 14mm (10g disk) or less than 10 mm (6g disk)). Litmus reduction test Principle: Litmus milk is a differential medium used to determine several metabolic functions of an organism :-(1) lactose fermentation, (2) caseolysis, and (3) casein coagulation. Litmus incorporated in milk is both a PH indicator and an oxidation reduction indicator which makes the medium capable of denoting several metabolic functions. Milk alone contains the carbohydrate lactose along with three main proteins: casein, lactalbumin, and lactoglobulin. Therefore, an organism may exhibit one or several metabolic properties in litmus milk, each specific for a particular species, aiding in bacterial identification. Litmus milk reduction test is a rapid technique to assist in the identification of enterococci. It is based on the ability of strains of enterococci to reduce litmus by enzyme action, shown by a decolorization of the litmus. Materials: - Litmus milk medium -Entercoccus strain -Viridans Streptococcus Procedure: 1. Using a large sterile loop, inoculate 0.5ml of sterile litmus milk medium with the test organism 2. Incubate at 35-37 C for up to 4 hours, examining at half hour intervals for a reduction reaction as shown by a change in color from mauve to white or pale yellow Result: - Enterococcus strain - Positive Viridans Streptococcus - Negative Note: - The incubation time should not be more than 4 hours because some strains of Viridans Streptococci will reduce litmus milk with prolonged incubation. CAMP test Principle: CAMP is an acronym for those individuals (Christie, Atkins, and Munch Peterson) who first described the phenomena of synergistic hemolysis between group B Streptococcus and beta-hemolytic Staphylococcus. The CAMP factor is diffusiable, protein like compound produced by Streptococcus agalactiae. A characteristic arrowhead hemolytic pattern results when the organism is streaked perpendicularly to beta-hemolytic S. aureus. Materials: 5% sheep blood agar Beta-hemolytic S. aureus Procedure: -

1. Streak S. aureus down the center of the blood agar plate 2. At a right angle to the S. aureus, streak 18 to24 hours culture to be tested. The two lines must not touch,
even after incubation.

3. Incubate at 35-37 C for 24 hours. Do not incubate an aerobically or under increased carbondioxide. False
positive result may occur. Result: Positive- A zone of enhanced hemolysis given by an arrowhead appearance at the junction of the Staphylococcus and streptococcus indicates the presence of group B Streptococcus (Example- S. agalactiae) Negative- No zone of enhanced hemolysis; Not group B Streptococcus (Example- S. bovis) Culturing of Gram-negative bacteria I. MacConkey agar

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Purpose: - MacConkey agar selects Gram-negative bacteria and also differentiates lactose fermenters (pink-red) from non-lactose fermenters (colorless). Principle: - The bile salts inhibit Gram-positive bacteria, which allows for the isolation of Gram-negative bacteria. Neutral red and crystal violet further inhibit the Gram-positive bacteria. Lactose is the only carbohydrate source. Neutral red indicator is brown in pH 6.8 to 8.0 and pink-red at pH less than 6.8. Procedure: 1. Prepare a test organism a clinical specimen aseptically 2. Streak on MacConkey agar for isolation 3. Incubate at 35-37 oC for 18 to 24 hours and observe for growth and color Result: If lactose is fermented, the medium is acidified, and bile salts are precipitated. The precipitated dye is observed, resulting in a pink-to-pink complex. II. Eosin-methylene blue (EMB) agar Purpose: - The EMB medium selects Gram-negative bacteria and also differentiates lactose fermenters (purple color to green metallic sheen) from non-lactose fermenters (colorless). Principle: - Eosin and Methylene blue are dyes that inhibit the Gram-positive bacteria. Lactose is the only carbohydrate source in most formulations. Procedure: 1. Prepare a test organism or clinical specimen aseptically 2. Streak on EMB agar for isolation 3. Incubate at 35-37 oC for 18 to 24 hours and observe for growth and color Result: - If lactose is fermented, precipitated eosin and methylene blue are observed, resulting in a purple color in the medium. E.coli produces a classical green metallic sheen, which is a rapid lactose fermenter. Non-lactose fermenters produce colorless colonies on EMB. III. Salmonella-shigella (SS) agar Purpose: - SS medium provides for inhibition of normal flora coliforms and differentiation of stool pathogens. Principle: - Bile salts inhibit Gram-positive bacteria, and brilliant green agar and bile salts inhibit the Gram-negative coliforms. Lactose is the sole carbohydrate source.Neutral red indicator is red in acidic conditions. Lactose fermenters appear pink-red, where as non-lactose fermenters clear. To detect hydrogen sulfide (H2S) production, sodium thiosulfate serves as a sulfur source. When H2S is formed, it combines with ferric ammonium citrate to form ferric sulfide (FeS), which is represented by black centered colonies. Procedure: 1. Prepare a test organism or clinical specimen aseptically 2. Streak on SS agar for isolation 3. Incubate at 35-37 oC for 18 to 24 hours and observe for growth and color Result: -The normal flora of coliforms appears pink-to-red colonies. Shigella appears as colorless colonies without black centers. Salmonella appears as colorless colonies with black centers. IV. Triple Sugar Iron (TSI) agar Purpose: - TSI agar can be considered an initial step in the identification of Enterobacteriaceae. Principle: -the medium contains protein sources (beef extract, peptone, yeast extract, peptone) that permit the growth of most bacterial strains, and glucose are present as well as phenol red indicator. Glucose is in a concentration one-tenth that of the other carbohydrates. Ferrous sulfate is present as an indicator of H2S production. The TSI is a two-reaction chamber with an aerobic slant portion and an anaerobic deep portion. The slant portion of the tube is exposed to the atmospheric oxygen and will become alkaline due to oxidative decarboxylation of peptides and amino acids. The slant tends to be come and remain alkaline (red). Amino acid degradation is minimal in the deep (anaerobic) portion, and thus a small quantity of acid produced can be detected because few amines are being formed from amino acids. Bacteria that ferment glucose, but not lactose or sucrose, only produce small quantities of acid and cannot counteract the degradation of amino acids at the slant, which results in an alkaline pH due to oxidative decarboxylation. Such organisms characteristically produce an alkaline slant over an acid deep (K/A). Organisms that ferment glucose and lactose and/or sucrose produce large quantities of acid, which overcome the alkaline reaction of the slant, yielding an acid slant over an acid deep (A/A). An organism in capable of fermenting glucose produces no changes in the indicator and is characterized by an alkaline slant over an alkaline deep (K/K). A sulfur source, sodium thiosulfate, provides sulfur atoms to detect the production of H2S gas. Hydrogen sulfide reacts with iron salts (ferrous sulfate or ferric ammonium citrate) to produce the black precipitate of ferrous sulfide. The

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presence of gas during fermentation is indicated by the presence of cracks in the medium or the pulling away of the medium from the walls of the test tube. Materials: Selected members of Enterobacteriaceae Non- fermentative Gram-negative Bacilli TSI slants Procedure: 1. Use single, isolated 18-24 hours colony 2. Select colony with sterile needle and stab with in half inch of the bottom of the agar 3. Streak colony up slant 4. Leave cap on loosely and incubate at 35 to 37 oC for 18 to 24 hours 5. Read and Interpret results Result: A/A* H2S _ Those organisms which ferment glucose with acid and gas Lactose and /or sucrose with acid and gas Example- Escherichia, Klebsiella and Enterobacter K/A* H2S + Ferment glucose with acid and gas, Lactose and sucrose not fermented Examples- Salmonella, Proteus, Citrobacter K/A H2S _ Glucose with acid; No gas. Lactose or Sucrose not fermented Example- Shigella, Serratia K/K H2S _Glucose not fermented. Lactose or sucrose not fermented Example- Pseudomonas, Alkaigenes A/A* H2S + Glucose fermented with gas; lactose or sucrose fermented. Example- Citrobacter freundii Note: A = Acid; A* = Acid and Gas; K= Alkaline Indole test Principle: Indole broth is used for distinguishing Enterobacteriaceae based on the ability to produce indole from tryptophan. The test is particularly useful for the identification of lactose fermenting members of Enterobacteriaceae. Escherichia coli is indole positive, whereas Enterobacter and Klebsiella are indole negative. Indole is also useful in the speciaton of Proteus. P.mirabilis is indole negative ,whereas P.vulgaris is positive. Tryptophan present in peptone is oxidized by certain bacteria to indole, Skatole, and indole acetic acid. The intracellular enzymes that metabolize tryptophan are known as tryptophanase. Indole is detected in broth cultures of bacteria with an aldehyde to form a red product. Two reagents may be used to detect indole, Kovacs and Ehrlichs. Tryptophan TryptophanaseIndole + Pyrovate + Ammonia Indole + P- Dimethylaminobenzaldehyde Red - Media and reagents - Tryptophan (1%) broth ( Commercial peptone, pancreatic enzymatic casein hydrolysate, or tryptone broths contain enough tryptophan) - Kovacs or Ehrlichs reagent Xylene or chloroform for extraction if using Ehrlichs reagent Procedure 1. Inoculate test organism on indole broth 2. Replace cap loosely and incubate at 35 oC for 18 to 24 hours 3. Add five drops of Kovacs reagent directly to the broth culture.Observe the red colour in the upper alcohol color. 4. If using Ehrlichs reagent, first add 1mlxylene or chloroform to the broth culture. Shake gently and then add five drops of reagent. Result Negative reaction: - no color development Positive reaction: - red ring at the interface of reagent or broth Variable reaction: - Orange color, indicates production of Skatole, a methylated intermediate that may be a precursor to indole production. VI. Methyl red-Voges- proskauer(MR-VP) tests Principle: MR-VP broth is a dextrose broth medium buffered with peptone. Glucose is fermented to pyruvic acid by one of two pathways, which results in either a positive MR or a positive VP test. The tests are particularly useful for the lactose fermenting Enterobacteriaceae.

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Media and reagents MR-VP broth: Glucose base MR pH indicator 5% alpha-naphthol in absolute methyl alcohol 40% KOH containing 0.3%creatinine Procedure 1. Inoculate medium with a heavy suspension of an 18 to 24 hrs culture 2. Incubate for 48 hours until sufficient growth occurs in broth. 3. After 48 hours, split broth by pipetting half in to a clean test tube. 4.i) perform MR test on one tube: a) add 5 drops of MR indicator to the aliquot with a Pasteur pipette b) Interpret color result immediately ii) Perform the VP test for the second aliquot a) add 0.6ml (6drops) of alpha-naphthol reagent to VP aliquot and shake well b) add 0.2ml (2drops) of 40% KOHG reagent to aliquot c) gently shake tubes for 30 seconds to 1 minute to expose reaction to atmospheric oxygen. This oxidizes acetoin to obtain a color reaction. d) Allow tubes to stand at least 10 to 15 minutes before attempting to interpret color results, although the reaction is often immediate. Note- the order of adding reagents is very important. A reversal of the order may lead to false negative results Result Positive MR test: - Distinct red color at surface of the medium eg. E. coli Negative MR test: - Yellow color at the surface of the medium eg. Enterobacter cloacae Positive VP test:- Pink red color at the surface of the medium eg. Enterobacter cloacae Negative VP test: - Yellow color at surface of the medium eg. E. coli Antimicrobial sensitivity testing Antimicrobial drugs include antibiotics and chemical antimicrobials (chemotherapeutic agents). Antibiotics are antimicrobial substances produced by living microorganisms. Antimicrobials are generally described as bacteriostatic when, at usual dosages, they prevent the active multiplication of bacteria, for example chloroamphenicol, tetracycline, and erythromycin. Antimicrobials are described as bactericidal when, at usual dosages, they kill bacteria, for example penicillins, Cephalosprins, polymyxins, and aminoglycosides. Some bacteriosatic antimicrobials become bactericidal when used at higher concentrations, for example erythromycin and tetracycline. Sensitivity testing is used to select effective antimicrobial drugs. Laboratory antimicrobial sensitivity testing can be performed using: A) Disk Diffusion test B) Dilution broth susceptibility test 4:1. Disk Diffusion test A standard suspention of organism is inoculated on to Muller-Hinton agar. Paper disks impregnated with specific antibiotic concentrations are placed on to the agar. After 18-24 hours of incubation the diameters of the zones of growth are measured. The results are compared with established values to determine the organisms Susceptable, Intermidiate or Resistance to each antibiotic. Objective: - To identify the susceptibility or resistance of pathogenic organisms to various antibiotics Reagents and Materials Muller-Hinton agar Susceptibility disks Turbidity standards I. Procedure for preparing turbidity standards 1.Prepare a 0.5 Mc Farland turbidity standard to measure inoculum density. Add 0.5 ml of 0.048N BaCl2 (or 0.5ml of 1.175% BCl2.2H2O) to 99.5ml of 0.36N H2SO4 (1%V/V). 2.Verify density of standard. The absorbance of a 0.5 McFarland turbidity standard at 625nm should be 0.08 to 0.1 with a 1cm path way, using matched cuvettes in the spectrophotometer. 3. Distribute 4 to 6ml amounts in to screw-cup tubes of the same size being used to grow the broth culture inoculum. 4. Seal tubes tightly and stored at room temperature in the dark.

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5. Vigorously agitate the turbidity standard using a vortex before each use. 6. Prepare a new standard at least every 3 to 6 months. II. Procedure for sensitivity test 1. Inoculate test plates by transferring pure colonies from the primary isolation medium in to approximately 5ml trypticase soy broth. 2. If the culture suspension is less turbid than the turbidity standard when read using adequate light against a white back ground with black lines, incubate the culture at 35c for 2 to 8 hours until the turbidity equals or exceeds that of the standard. Dilute the culture suspension with the sterile saline if necessary. Proceed with out incubation if the turbidity of the inoculated suspension is comparable to that of the standard. 3. Dip a sterile cotton swab in to the standardized culture suspension and express fluid by rotating the swab firmly against the inside wall of the test tube. 4. Inoculate the entire agar surface, streaking in three different directions by rotating the plate at 60-degree angles after each streak. If the plate is properly streaked, a confluent lawn of growth will develop. Avoid extremes of inoculum density. 5. Allow inoculum to dry 3 to 15 minutes with top in place. 6. Apply appropriate susceptibility disks manually or with dispenser on to the inoculated agar surface. Gently press each disk down with sterile forceps, flame, and cool between each disk to ensure complete contact with the agar. A minimum spacing of 24mm from disk center to center to avoid over lapping inhibition zones. Do not move a disk once it has contacted the agar, since the antibiotics diffuse almost in stantaneously. 7. Incubate the inverted plates at 35 c for 24 hours. 8. Measure diameters of complete inhibition to the nearest whole millimeter using caliper or ruler. 9. Interpret the size of zones of inhibition by using zone diameter interpretative standards. 10. Report the result of each antibiotic as susceptible (S), intermediate (I) or resistant (R). 4:2. Dilution broth susceptibility test Objectives: - To determine the MIC and MBC of a particular antibiotics Principle: Dilutions of an antibiotic are added to Muller-Hinton broth and a standard concentration of the inoculum. The tubes are incubated overnight and read usually for turbidity. The lowest concentration showing no visible growth or microscopic inhibition is the minimum inhibitory concentration (MIC). Broth culture showing no visible growth are subcultured on to appropriate agar and incubated to determine if the organism has been killed. The lowest concentration of antibiotic that exhibits at least 99.9% killing on sub-culture is the minimum bactericidal concentration (MBC). Materials: - Antibiotic standard- for penicillin uses a working solution of 20U of antibiotics, use a 200mg/ml working solution. - Over night culture grown at 35 0C on blood agar. - Trypticase soy broth with over night culture - Mueller-Hinton (MH) broth - Sterile 13X10 mm test tubes with screw caps Procedure: 1. Set up 13 sterile test tubes with caps and level 1 through13. Tube 11 will be the inoculum control Tube 12 will be the broth control Tube 13 will be the antibiotic control 1. Pipet 1ml of Mueller-Hinton broth in to tube 2 through 10, tube 11 & tube 13. Pipet 2ml of MH broth in to tube 12. 2. Pipet 1ml of the working antibiotic solution in to tubes 1, 2 & 13. 3. Make two fold dilutions in 1ml amounts in tubes 2 through 10. Mix & change pipettes at each dilution. This is done by mixing tube 2 and transferring 1 ml of its contents to tube 3. The process is continued to tube 10. Discard 1ml from tube 10. 4. Standardize the inoculum to a 0.5 Mc Farland standard. Then further adjust the inoculum by adding 0.1ml of the inoculum to 19.9ml of MH broth. This is a 1:200 dilution. The final concentration of bacteria is approximately 1X105 to 1X106 CFU/ml. 5. Pipet 1 ml of the standardized inoculum in to tube 1 through 11. Mix each tube well and incubate at 37Co for 18 to 24 hours. 6. To determine MBC and MIC sub culture 0.01ml from each tube showing no visible growth on to blood agar plate. Incubate at 35 to 37 Co for 18 to 24 hours. activity/ ml; for other

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Result: Determination of MIC: - The tube with the lowest antibiotic concentration showing no visible growth is considered to be the MIC. Tube 11- Must show visible growth for test tube to be valid (inoculum control). Tube 12- Must show no visible growth for test tube to be valid (both control) Tube 13- Must show no visible growth for test tube to be valid Antibiotic control) MBC is the lowest concentration that results in 99.9% killing. Bacterial Resistance to Antimicrobial Agents: Enzymatic Principle: The activity of antimicrobial agents is usually very specific, affecting primarily essential bacterial cell structures or biochemical processes. Resistance to antimicrobial agents can result from a mutation in a gene on the bacterial chromosome, or by acquisition from another organism of a plasmid (extrachromosomal DNA) that bears one or more resistance genes (R-factor). Acquisition of an R-factor can suddenly render a previously susceptible bacterium resistant to multiple antimicrobial agents. One of the most common mechanisms of bacterial resistance is the production of specific enzymes that destroy antimicrobial agents before they can affect the bacterium. Routinely, the clinical microbiology laboratory tests for bacterial susceptibility or resistance by the methods described in above Experiments. Alternatively, however, if you are interested only in the response of a given organism to a particular antimicrobial agent (e.g., Neisseria gonorrhoeae to penicillin), you can test the organism for its ability to produce a sufficient amount of an enzyme that specifically inactivates that drug. One such test is illustrated in the following experiment, using a penicillin susceptible organism and one that is resistant to penicillin because it produces penicillinase. Purpose To detect penicillinase production by a test bacterial strain Materials Filter paper disks impregnated with nitrocefin for performing the beta-lactamase test Sterile water or saline Clean glass slides Forceps Plate culture of a penicillin-resistant Staphylococcus aureus Plate culture of a penicillin-susceptible Bacillus subtilis Procedures 1. Place two small drops of water or saline on the surface of a clean glass slide. 2. Pick up a beta-lactamase disk with your forceps and place it in contact with one drop of fluid. 3. Repeat this procedure with a second disk, placing it on the second drop of fluid. Do not oversaturate the disks. 4. With your sterilized and cooled inoculating loop, pick up a portion of a B. subtilis colony and rub it across the surface of the first disk. 5. Rub a portion of a S. aureus colony across the surface of the second disk. 6. Observe the areas on the beta-lactamase disks where the organisms were inoculated for up to 30 minutes. A positive result is usually seen within 3 to 4 minutes. Results 1. A change in the color of the bacterial growth rubbed on the disk from yellow to red is a positive test indicating degradation of nitrocefin. 2. Record your results in the chart. Bacterial Resistance to Antimicrobial Agents: Mutation Principle: In Experiment above, you detected bacterial resistance resulting from an enzyme whose production was directed by a gene on a bacterial plasmid. In this experiment, you will detect resistance resulting from a mutation in a gene on the bacterial chromosome. Most bacterial mutations go unrecognized and the mutants may not survive unless the mutation provides a selective advantage for the cell, such as the ability to survive in the presence of an antimicrobial agent that is lethal for the wild-type (non-mutated) population. Streptomycin is an antimicrobial agent that, like gentamicin, inhibits protein synthesis by acting on the bacterial ribosome. E. coli organisms become resistant to streptomycin with just one mutation so that, as we shall see, this organism antimicrobial agent combination is convenient to use to illustrate the mechanism of chromosomal resistance. Purpose To select E. coli mutants resistant to streptomycin Materials: Tubes containing 19 ml of nutrient agar (first session) Tubes containing 20 ml of nutrient agar (second session) Flasks containing 49 ml of nutrient broth Sterile petri plates Streptomycin solution (20 mg/ml) 18- to 24-hour broth culture of Escherichia coli 1-ml pipettes Tubes contining 0.5 ml sterile water

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Procedures 1. During this experiment, you will have to perform several steps quickly before the agar in the tubes solidifies. 2. Place 3 melted tubes of nutrient agar into a 50C water bath. 3. With your marking pen or pencil, label one tube of melted agar, one flask of broth, and one petri plate SM 100 (that is, with 100 _g of streptomycin/ml). Label another tube of melted agar, one flask of broth, and one petri plate SM 250 (with 250 _g of streptomycin/ml). Label the third tube of melted agar, one flask of broth, and one petri plate No SM (without streptomycin). The last tube and flask serve as your controls. 4. Add 0.1 ml of the streptomycin solution to the melted tube labeled SM 100 and 0.25 ml streptomycin to the tube labeled SM 250. 5. Immediately add 1 ml of the overnight culture of E. coli to each agar tube containing streptomycin and the control tube without streptomycin. Mix well by rotating each tube between the palms of your hands (not by shaking) and quickly pour into the petri plates labeled to correspond to the tubes. Set the plates aside to harden. 6. Add 0.25 ml of the streptomycin solution to the flask of broth labeled SM 100 and 0.6 ml to the flask labeled SM 250. 7. Add 1 ml of the E. coli culture to each flask with streptomycin and the control flask without streptomycin. 8. Once the plates have hardened (step 5), seal around their edges with strips of Parafilm as demonstrated by the instructor. Incubate all plates and flasks at 35C. At the next session, you will determine whether colonies resistant to streptomycin have arisen (steps 9 through 18). 9. Label two tubes of melted nutrient agar SM100, label two tubes SM 250, and two tubes No SM. Place all tubes in a 50C water bath. 10. Label the bottoms of six petri dishes as follows (refer to the figure):agar SM 100,agar SM 250,agar No SM,broth SM 100,broth SM 250,broth No SM.Now divide each of the 6 plates into three sections with your marking pen or pencil. Label the sections of each plate as follows:SM 100,SM 250,No SM. 11. To each of the two tubes of melted agar (step 9) labeled SM 100, add 0.1 ml of the streptomycin solution. Pour one into the petri plate labeled agar SM 100 and one into the plate labeled broth SM 100. 12. To each of the two tubes of melted agar labeled SM 250, add 0.25 ml of the streptomycin solution and pour into the appropriate plates labeled SM 250 as in step 11. 13. Pour the last two tubes of agar without streptomycin into the corresponding petri plates. Set plates aside to harden. 14. Examine the broths and plates inoculated at the previous session. 15. Record your observations of growth or no growth in the chart. 16. From each agar plate on which you see bacterial growth, suspend a few colonies in a tube of sterile water. Streak a loopful of the suspension onto the correct section of the 3 freshly prepared agar plates labeled agar. For example, colonies growing on the plate that contained 100 g of streptomycin/ml, will be streaked onto the three sectors labeled SM 100 on each plate. 17. Streak a loopful from each of the three flasks onto the corresponding sectors of the plates labeled broth. 18. Seal all plates with Parafilm and incubate at 35C. 19. Examine the plates at the next session and record your results in the chart.

MYCOLOGY 1. Wet mount preparation Refer experiment No. 6.1 2. KOH Preparation Objective: To investigate fungal infections. Principle: - Fungi are usually larger than bacteria, and in material from skin, hair or nails, they can be seen by direct microscopy provided the material is first softened and cleared with a strong alkali such as 20% w/v potassium hydroxide (KOH). The purpose of the alkali is to digest the keratin surrounding the fungi so that the hyphae and conidia (spores) can be seen. Materials required: - Slide - Cover glass - Surgical blade - Applicator stick - Petridish - Cotton - 20% w/v KOH - Pasture pipette - Microscope Procedure:

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Place a drop of KOH (20%) solution on a slide. Transfer the specimen (small pieces) to the drop of KOH, and Cover with cover glass. Place the slide in a petridish or other container with a lid together with a damp piece of cotton wool to prevent the preparation from drying out. Hairs clear within 5-10 min. Skin scales and crusts clear within 20-30min. Pieces of nail, may take several hours to clear. As soon as the specimen has cleared, examine it microscopically using the 10x and 40x objectives.

Result: Observe fungal elements (the hyphae and spores) In unstained preparations, since the refractive index of the bacterium is more or less the same as the background (fluid) it is not easy to detect it. 3. India Ink Stain Purpose: The procedure is applicable only to suspected positive Crytococcal cultures. Procedure For suspected Crytococcal cultures, make a wet preparation using saline on a clean glass slide, then add a small drop of India Ink and mix. Apply a large coverslip ((22 x 40 mm) over the mixture and press it gently to obtain a thin mount. If India Ink is too thick (dark), dilute it by 50% with saline. Allow the preparation to stand for few minutes to settle. Scan under low power in reduced light. Switch to high power if necessary. Interpretation The mucoid capsule appears as a clear halo that surrounds the yeast cell or lies between the cell wall and the surrounding black mass of India Ink particles. Capsules may be broad or narrow. The yeast cells may be round, oval or elongate. Buds may be absent, single or rarely multiple and may be detached from the mother cell but enclosed in a common capsule attached. 4. Lactophenol Aniline Blue (LPAB) Principle: LPAB contains lactic acid as a clearing agent, phenol as a disinfectant, glycerol to prevent drying and Aniline Blue which is the dye that stains fungi. LPAB is a wet preparation Purpose: To determine the morphology of the conidiogenous cells and the conidia that they give rise to in order to identify a filamentous fungus. Procedure: 1) Tease prep The test must be performed in the Laminar Airflow Biosafety Cabinet. First, observe the gross morphology of the colony carefully to determine whether or not the culture is mouldy, granular or a mixture of both. It is important to prepare the LPAB preparation by "teasing" the fungus not "chopping" it. Materials required for LPAB staining: 1. LPAB reagent 2. Probe to get the specimen 3. Teasing needles 4. Glass slides 5. Coverslips 6. Lead or wax pencils 7. Disinfectant bucket 8. Electric incinerator 9. Clear nail polish 10. Slide tray 1) Tease Prep Procedure: Sterilize the loop and the needles in the incinerator and allow them to cool. Label slide, place 2 drops of LPAB reagent on the slide. Cut a small piece of the fungus from a granular or colored part of the colony, somewhere away from the central part towards the periphery and place the piece of the fungus in the LPAB in the upside down position. Hold the thallus with the needle and gently tease the inverted side of the specimen into the staining (LPAB) fluid.

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After enough teasing, remove all the solid particles and the agar from the mixture and discard in the disinfectant container. Put a coverslip gently onto the LPAB preparation and hold the slide preparation briefly over the incinerator opening. Heating the slide will help to stain the cell wall of the fungi and kill the spores on the surface of the slide. Seal the preparation with nail polish if necessary for a permanent mount. Examine under the light microscope using the low power objective.

2) Scotch tape prep from plate culture (Not done on suspected dimorphic fungi) 1. Place a drop of LPAB onto a clean glass slide. 2. Take a small piece of clear scotch tape and loop back on itself with sticky sides out. 3. Hold the tip of the loop securely with forceps. 4. Press the sticky side firmly to the surface of the fungal colony. 5. Pull the tape gently away from the colony. 6. Open up the tape strip and place it on the drop of LPAB on the glass slide, making sure that the entire sticky side adheres to the slide. 7. Examine under the light microscope. 6. Calcofluor white stain Principle: Calcofluor White stain is useful for staining skin scrapings, hair, nail and thick tissue specimens. Calcofluor staining requires the addition of KOH which helps to dissolve keratinized particles and helps to emulsify solid, viscous material that may mask the fungal elements. Calcofluor method is NOT suitable for the detection of Pneumocystis carinii. Calcofluor stained smears are read under the UV microscope as for the Fungi-Fluor stain. Purpose: To identify fungi from scrapings, hair, nail and thick tissue specimens using calcofluor white stain Requirements: Glass slides KOH Calcofluor white stain Fluorescent microscope Procedure: Place a portion of the specimen on the slide (select a purulent area if secretions). Add 1 to 2 drops KOH and emulsify specimen. If it does not clear rapidly, place slide in petri dish and allow to stand about 10 minutes. If tissue or scrapings, place the slide on 35oC bench top heating block for 15-20 minutes to speed clearing. Add 1 or 2 drops of calcofluor white reagent and mix thoroughly. Calcofluor white may be added right after KOH. Apply coverslip gently. Make sure specimen does not overflow. Examine under fluorescent microscope Always include a control slide positive for yeast or filamentous fungus. NB: In the event when no Fungal Stain smear is available, Gram smear may be retrieved for over staining by Calcofluor White or KOH. Interpretation Fungal cell walls fluoresce apple green (see Fungi-Fluor stain). 7. Fungi-Fluor Stain Principle: The active, colourless, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of 4,4bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-triazin-2-ylamino]-2,2'-stilbenedisulfonic acid. Fungi-Fluor staining solution is a 0.05% solution of this dye in deionized water with potassium hydroxide added as a clearing agent. The Fungi-Fluor counter staining solution B is an aqueous solution of Evans Blue dye used to reduce background fluorescence. Cellufluor binds nonspecifically to beta-linked polysaccharides found in chitin and cellulose which are present in fungal cell walls. When exposed to long wave UV light, fungal cell walls will fluoresce. Purpose: The Fungi-Fluor stain is used for the rapid identification of various fungal elements in fresh or frozen clinical specimens. NB: Collagen, elastin, cotton fibres, plant material, some cells, cell inclusions and parasite cyst forms (e.g. Acanthamoeba) may fluoresce. Requirements Staining Solution A Counterstaining Solution B

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Absolute alcohol Water Fluorescent Microscope Precautions Store in a dark or opaque bottle, tightly sealed, at room temperature. Avoid eye or skin contact: use gloves and protective glasses. Procedure Prepare smear of specimen and allow to air dry. Fix on the rack with absolute methanol for 5 minutes until dry. Fixed smears can be held indefinitely until ready to stain and examine. Add a few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute. Rinse gently with tap water. Apply coverslip to wetted slide and examine with the fluorescent microscope using the designated filter. If there is a delay, add fresh distilled water to the coverslip just prior to examination. Optional for thicker smears. Add few drops of the counterstain Fungi-Fluor solution B. Rinse gently with tap water and then proceed as in step 5 above. NB: Gram stained smears can be overstained with Fungi-Fluor after removing immersion oil with alcohol. Similarly, Fung-Fluor stained slides may be overstained with other stains such as GMS, PAS, Geimsa, etc.

Interpretation Use 40x objective Fungal cell walls will fluoresce apple-green. Observe for characteristic morphology to differentiate from artifacts and background. When the counterstain is used, fungi will appear yellow-green against a red-orange background. 8. Germ Tube Test Principle: This is a rapid test for the presumptive identification of C. albicans. Reagents / Materials / Media Bovine serum - A small volume to be used as a working solution may be stored at 2 to 8 oC. Stock solution can be dispensed into small tubes and stored at -20oC. Clean glass microscope slides Glass coverslips Glass tubes (13 x 100 mm) Pasteur pipettes Procedure Put 3 drops of serum into a small glass tube. Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum. The pipette can be left in the tube. Incubate at 35oC to 37oC for up to 3 hours but no longer. Transfer a drop of the serum to a slide for examination. Coverslip and examine microscopically using x 40 objective. Interpretation Germ tubes are appendages half the width and 3 to 4 times the length of the yeast cell from which they arise. There is no constriction between the yeast cell and the germination tube. Positive test: presence of short lateral filaments (germ tubes) one piece structure Negative test: yeast cells only (or with pseudohyphae) always two pieces 9. Slide culture Purpose: To carry out slide culture procedure Principle: A slide culture is used to preserve and observe the natural state and the actual structure showing conidiogenous cells and conidia of the fungus. Before setting up a slide culture, it is important to do an LPAB preparation and demonstrate that the organism is susceptible to Cycloheximide (Exception: Dermatophytes and Sporothrix schenckii). Procedure 1. Place a sterile filter paper in a small petri dish. 2. Add some sterile distilled water. 3. Place 2 pieces of wooden sticks on the filter paper, a few centimetres apart.

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Place a clean alcohol flamed heat sterilised glass slide on the sticks. Using a sterile spatula, cut a small square (about 1 x 1 cm) of agar block from the Potato Dextrose Agar (PDA) (or 3% Salt Agar) and place it on the centre of the slide. 6. Using a needle or spatula, inoculate the agar with a small amount of fungus under test on each of the four sides of the block. 7. Place a heat-sterilized coverslip over the block and press down gently. 8. Incubate at 28oC or room temperature. 9. Examine daily for sufficient growth. 10. When good growth appears, place a drop of LPAB on a clean slide, remove the coverslip using forceps, pass the top side of the coverslip in front of the incinerator opening to fix and place it over the LPAB on the slide. 11. Examine under the light microscope. If the preparation shows sufficiently developed structures, prepare the second preparation from the slide by removing the agar block from the slide. Discard the agar in the sharps container. Place a drop of LPAB on the slide and a new coverslip on it. Both preparations can be preserved indefinitely by sealing the edges with nail polish. 12. If the fungus is still underdeveloped, add a fresh coverslip on the agar block, reseal the plate with parafilm and continue incubation.

4. 5.

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