MCB 1000L

Applied Microbiology Laboratory Manual

By Frances Duncan With contributions from Valerie Walker and Neil Clark Fourth Edition 2005

Table of Contents Topic Safety Aseptic Technique Microscopy Smears Staining Culturing and Isolation Techniques Staphylococcus Streptococcus Throat Culture Oxidase Urea Triple Sugar Iron Agar (TSI) Motility IMViC Disc Diffusion Susceptibility Methods Guidelines for Identification of Unknowns References Page Number 3 8 11 17 20 25 33 38 45 48 50 52 56 58 61 67 70

Safety
Introduction In the laboratory individuals are exposed to hazards not found in a regular classroom. It is essential that students follow all rules established by the lab instructor, lab manager, or lab assistant to ensure the safety of all individuals in the class. Failure to follow established rules may result in dismissal of the individual from the class. Laboratories have certain standard safety equipment. These typically include a general-purpose fire extinguisher, eyewash, safety shower and cut off switches for electrical and gas outlets. It is the responsibility of the student to locate and know how to use the general safety equipment in the laboratory. Additionally, students should be aware of exits from the room in case of emergency, the location of the nearest fire call box, how to summon Campus Security, and how to obtain emergency medical assistance. The microbiology lab has some additional safety considerations. Since individuals work with potentially pathogenic organisms care must be taken to prevent possible infection or transmission of the organisms from the laboratory. Students must wear protective clothing (lab coats) while working the laboratory. Lab coats may not be worn outside the laboratory. Intact skin is an adequate barrier against microorganisms so gloves are not necessary in lab. Gloves will be provided and students may wear gloves when handling cultures if they so desire. Tabletops must be disinfected before and after lab using the disinfectant provided. Instruction in aseptic technique will be provided. Aseptic technique must be followed while working with microorganisms. Handwashing is a simple and effective way to prevent the transmission of disease. While antibacterial soap may provide some additional protection the major effect of handwashing is the mechanical removal of microbes from the skin. Friction when washing hands is important to mechanically remove organisms from the surface of the skin. Using a paper towel to turn off the water prevents recontamination of the hands with microorganisms. Hands must be washed whenever the student leaves the lab. Two copies of the Laboratory Safety Rules are included. One must be signed and returned to the laboratory instructor at the end of class. The additional copy is for your reference.

Microbiology Laboratory Safety Rules

1. All materials and clothes other than those needed for the laboratory are to be kept away from the work area. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be worn outside of the laboratory. Clean the lab table before and after lab with the disinfectant solution provided Wash hands before leaving lab. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable items are to be placed in the BIOHAZARD container. Reusable items are to be placed in the designated area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate container Reusable items should have all tape and marks removed by the student before being autoclaved. Because organisms used in this class are potentially pathogenic, aseptic technique must be observed at all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouth pipetting is not allowed. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on request. Long hair should be tied back while in lab. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructor immediately. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards. o o Bacticinerators reach an internal temperature of 850 C or 1500 F. Keep all combustibles away from the Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator. Microscopes and other instruments are to be cared for as directed by the instructor. It is the responsibility of the student to know the location and use of all safety equipment in the lab (eyewash, fire extinguisher, etc.) Cultures may not be removed from the lab. Visitors are not allowed in the lab. Doors and windows are to be kept closed at all times. 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.

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I have read and understand the above rules and agree to follow them. Signed_____________________________________________ Date_________________ Name (Please print)________________________________________________________

Microbiology Laboratory Safety Rules

1. All materials and clothes other than those needed for the laboratory are to be kept away from the work area. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be worn outside of the laboratory. Clean the lab table before and after lab with the disinfectant solution provided Wash hands before leaving lab. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable items are to be placed in the BIOHAZARD container. Reusable items are to be placed in the designated area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate container Reusable items should have all tape and marks removed by the student before being autoclaved. Because organisms used in this class are potentially pathogenic, aseptic technique must be observed at all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouth pipetting is not allowed. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on request. Long hair should be tied back while in lab.

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6. 7.

8. 9.

10. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructor immediately. 11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards. o o Bacticinerators reach an internal temperature of 850 C or 1500 F. Keep all combustibles away from the Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator. 12. Microscopes and other instruments are to be cared for as directed by the instructor. 13. It is the responsibility of the student to know the location and use of all safety equipment in the lab (eyewash, fire extinguisher, etc.) 14. Cultures may not be removed from the lab. Visitors are not allowed in the lab. 15. Doors and windows are to be kept closed at all times. 16. 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. I have read and understand the above rules and agree to follow them. Signed_____________________________________________ Date_________________ Name (Please print)________________________________________________________

Safety Review Questions 1. List all emergency exits from the laboratory.

2.

Describe how you would reach Campus Security.

3.

Describe how you would obtain emergency medical assistance.

4.

What protective clothing must be worn during lab?

5.

What is a simple and effective way to prevent disease transmission?

6.

What general safety equipment is found in the laboratory?

7.

Where is the nearest fire call box?

8.

What do you need to bring to the lab table for lab?

9.

How do you dispose of material that may be contaminated with bacteria?

10.

How do you dispose of a broken slide?

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11.

You have just disinfected your lab table. Where do you dispose of the paper towels you used?

12.

After washing your hands, where do you dispose of your paper towels?

13.

When discarding reusable contaminated material where do you put it? What must be done to it before it is discarded?

14.

It is the end of lab. What must you do before you leave lab? List the tasks in order of performance.

Aseptic Technique
Introduction When working with microorganisms it is desirable to work with a pure culture. A pure culture is composed of only one kind of microorganism. Occasionally a mixed culture is used. In a mixed culture there are two or more organisms that have distinct characteristics and can be separated easily. In either situation the organisms can be identified. When unwanted organisms are introduced into the culture they are known as contaminants. Aseptic technique is a method that prevents the introduction of unwanted organisms into an environment. When changing wound dressings aseptic technique is used to prevent possible infection. When working with microbial cultures aseptic technique is used to prevent introducing additional organisms into the culture. Microorganisms are everywhere in the environment. When dealing with microbial cultures it is necessary to handle them in such a way that environmental organisms do not get introduced into the culture. Microorganisms may be found on surfaces and floating in air currents. They may fall from objects suspended over a culture or swim in fluids. Aseptic technique prevents environmental organisms from entering a culture. Doors and windows are kept closed in the laboratory to prevent air currents which may cause microorganisms from surfaces to become airborne. Once these microbes are airborne they are more likely to get into cultures. Transfer loops and needles are sterilized before and after use in the Bacticinerator to prevent introduction of unwanted organisms. Agar plates are held in a manner that minimizes the exposure of the surface to the environment. When removing lids from tubes, lids are held in the hand and not placed on the countertop during the transfer of materials from one tube to another. These techniques are the basis of laboratory aseptic technique. In this laboratory exercise the location of environmental organisms will be explored and how microorganisms can be transmitted through contact with contaminated surfaces.

Laboratory Procedure General Instructions 1. Students will work in groups. Materials/Equipment 2 blood agar or nutrient agar plates per student plus one plate per group Markers Instructions 1. Label one plate Open. Write on the agar containing side of the plate, not on the lid. Remove the lid from the plate and place in on the lab table, agar side up, until the end of lab. 2. Obtain one agar plate per student and draw a line on the agar containing side of the plate to divide the plate in half. Label one side dirty and one side clean. Remove the lid and gently touch your fingertips to the agar on the dirty side. Replace the lid. Wash your hands or clean your hands with hand sanitizer and gently touch your fingertips to the agar on the clean side of the plate Obtain one agar plate per student and using a marker divide the plate into quadrants. Label the quadrants 1, 2, 3, and 4. Put on gloves and try to touch as few surfaces as possible. The lab instructor will swab the left gloved palm of each student. Remove the lid from your agar plate and touch the first two fingers of your right hand to the agar in quadrant 1. Replace the lid on the agar plate and touch the first two fingers of your right hand to the left palm of another student in your group. Remove the lid from your agar plate and touch the first two fingers of your right hand to the agar in quadrant 2. Repeat steps 6 and 7 with two other students in your group and inoculate quadrants 3 and 4. Carefully remove your gloves and place them in the biohazard container. Wash your hands. Replace the lid on the open plate. Stack all plates agar side up and incubate them until the next lab period. During the next lab period examine plates for growth and record results on page 10. Discard all plates in the biohazard container.
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Conclusions 1. Describe the growth on the plate labeled Open.

2.

Are organisms found in the air? What results support your conclusions?

3.

Record your results from the first plate you inoculated with your hands in the chart below. Side of Plate dirty clean Results

4.

What effect does hand washing have on microorganisms? Should you ever touch a sterile surface?

5.

Record the results from the second plate you inoculated with gloved hands in the chart below. Quadrant 1 2 3 4 Results

6.

One person in your group had microorganisms swabbed on their glove. The others did not. From you results can you determine who had the contaminated glove?

7.

What conclusions can you draw from your data concerning where microbes are found in the environment?

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Microscopy
Introduction Microorganisms are too small to be seen with the naked eye so a microscope must be used to visualize these organisms. While a microscope is not difficult to use it does require some practice to develop the skills necessary to use the microscope to its maximum capabilities. Bacteria and other cellular microorganisms are measured in micrometers (m) or 1 x 10-6 meters. Viruses are even smaller and are measured in nanometers (nm) or 1 x 10-9 m. When carrying a microscope always use both hands. One should be on the arm of the microscope and one should be under the base of the microscope. Discussion There are several types of microscopes but the only one used in this laboratory is the compound light or bright-field microscope. Individual microscopes will vary depending on the manufacturer but all microscopes have the same basic features. Ocular Nosepiece Arm Coarse Adjustment Knob Fine Adjustment Knob Light Source

Objectives Stage Iris Diaphragm Stage Adjustment Knob

Base

These microscopes are known as compound microscopes because there are two magnifying lenses in the microscope. One magnifying lens is in the ocular and one is in the objective. Each contributes to the magnification of the object on the stage. The total magnification of any set of lenses is determined by multiplying the magnification of the objective by the magnification of the ocular. The nosepiece rotates allowing the objectives to change and thus change the magnification of the microscope.

The stage is where the slide is placed. The stage adjustment knobs allow the slide to be moved easily. Light provides the illumination for the specimen. To control
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the amount of light reaching the eye the iris diaphragm may be opened or closed using the lever just under the stage. On low magnifications less light is need than on higher magnifications. Too much light on low magnification may mask the specimen, particularly something as small as a bacterial cell. The coarse and fine adjustment knobs are used to focus on the specimen. When a slide is on the stage there is a space between the objective and the slide. This space is known as the working distance. The coarse adjustment knob will cause the working distance to visibly change while the fine adjustment knob is for final, fine focusing. The ability to see things using a microscope is limited by the resolving power of the microscope. The resolving power of a microscope is the distance two objects must be apart and still be seen as separate and distinct. For the light microscope this is 0.2 m. Objects closer together than 0.2 m will not be distinctly seen. Increasing the magnification will not make the objects more distinct, just bigger. Each objective has the magnification of the objective written on the objective. The magnification of the ocular is also inscribed on the ocular. Low magnifications are used for quickly examining the slide to find an appropriate area to examine. Higher magnifications allow the examination of a particular object on the slide. Examine your microscope and complete the table below.

Objective
Scanning Low Power High Power Oil Immersion

Magnification of Magnification of Objective Ocular

Total Magnification

When you look through the ocular you will see a lighted circle. This is known as the field of view or the field. While looking through the microscope move the iris diaphragm lever and notice how the brightness of the light changes. As you move the objectives to provide increased magnification you will look at a smaller section of the slide. Be sure you move the object you want to view into the center of the field before moving to the next objective. These microscopes are parfocal. Once you have focused on an object using one objective the object will be approximately in focus on the next objective. Use of the fine focus knob will sharpen the focus.

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Procedure for Focusing 1. 2. 3. Obtain a slide. Use the coarse adjustment knob to obtain maximum working distance. Place the slide on the stage. The slide should fit into the slide holder but is not placed under the slide holder. Use the stage adjustment knob to move the slide over the hole in the stage. Rotate the low power (10X) objective in place. Use the coarse adjustment knob to obtain the minimum working distance. Develop the habit of watching this process to be sure the objective does not crash into the slide. Look through the ocular. Adjust the light with the iris diaphragm lever if necessary. Slowly turn the coarse adjustment knob until something comes into focus. Use the fine adjustment knob to sharpen the focus. Using the stage adjustment knob move the slide around until you find an area you wish to examine more closely. Move the slide until the object you wish to examine is in the center of the field. Rotate the high power objective into place. Use the fine adjustment knob to sharpen the focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever if necessary. Rotate the high power object halfway to the next position. Place a drop of immersion oil on the slide, then rotate the oil immersion objective into place. The objective should be immersed in the oil on the slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using the iris diaphragm lever if necessary. When finished viewing the slide use the coarse adjustment knob to maximize the working distance and remove the slide from the stage. If you want to look at another slide, begin the process over. If you are finished with the microscope clean the microscope and return it to storage.

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Procedure for Cleaning a Microscope 1. 2. Turn off the light and unplug the cord. Store the cord appropriately. Using the coarse adjustment knob to obtain maximum working distance and remove the slide from the stage. Using lens paper clean all the lenses starting with the cleanest firstocular, low power, high power and oil immersion. Use lens cleaner if necessary. Clean any oil off of the stage using Kimwipes or paper towels. Rotate the scanning objective into place. Use the coarse adjustment knob to obtain minimum working distance. Return the microscope to the appropriate storage area.

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4. 5.

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Review Questions Label the drawing of the microscope.

Define: Resolving power Parfocal Field Working distance Tell the function of each of the following. Coarse adjustment knob Fine adjustment knob Iris diaphragm Stage adjustment knob What unit of measurement is used for measuring bacteria?

How do you determine the total magnification of a set of lenses?

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Describe the process for focusing on a slide.

Describe how to properly clean a microscope.

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Smears
Introduction The microscopic examination of microorganisms is a valuable identification technique. In order to view microbes it is necessary to prepare slides of the organisms. Microscopic preparations may be either wet mounts or smears. Wet mounts involve placing cells in a drop of water, adding a coverslip and viewing the material under the microscope. In microbiology most of the organisms viewed are bacteria which are small and difficult to see without staining. Wet mounts are temporary preparations and the ability to stain is limited. A smear is a thin preparation of cells allowed to dry on a slide. This material is then fixed to the slide using heat or a chemical. A smear is a more permanent preparation and may be stained using a variety of techniques. Smears are made using plain tap water. While tap water is not sterile it has too few organisms in it to interfere with a bacterial smear. At least 500,000 cells per milliliter must be present in order to see one cell per oil immersion field. Bacteria are mixed in water and allowed to dry on the slide to make a bacterial smear. This is then fixed to the slide using heat. Heat fixing helps attach the cells to the slide so they are not washed off during the staining process, kills the cells so the slide is not hazardous to handle, and alters the cell wall for staining. The number of cells placed on the slide is important for viewing the cells. Too few organisms and it is ha rd to find them on the slide. Too many organisms and it is difficult to see individual cells to determine their morphology or shape. Laboratory Procedure General Instructions 1. 2. Students work individually. To sterilize an inoculating loop or needle insert the loop or needle into the Bacticinerator and observe it. It must glow red for three seconds to be sterilized. Loops and needles should never be propped in the Bacticinerator. The handles are aluminum and will melt. Also they conduct heat readily a nd can cause burns if the handles heat up. A hot loop or needle must cool slightly before touching a bacterial colony to prevent killing the cells. To aseptically remove a lid from a bottle or tube, grasp the lid with the little finger of the dominant ha nd. Twist the bottle or tube to loosen and remove the lid. Do not put the lid on the table but keep it in your hand while removing material from the bottle or tube. Return the lid to the bottle or tube by turning the bottle or tube to tighten the lid.

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Materials/Equipment Clean glass slides Prepared cultures of Staphylococcus aureus and E. coli Inoculating loop Bacticinerator Laboratory marker Instructions 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. Label two slides across one end Staph. and two slides E. coli. 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. Tap the loopful of water onto the center of one of the labeled slides. Sterilize the loop. 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. 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. 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. Repeat Steps 2 -8 to make two smears of Staphylococcus aureus and two smears of E. coli. Store the slides in slide boxes for use in future lab exercises.

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Smear Review Questions 1. Describe two preparations that may be used to observe microorganisms.

2.

What is the purpose of heat fixing?

3.

Outline the procedure for making a smear?

4.

Why must slides used in smear preparation be grease-free?

5.

Is it necessary to use sterile water when making a smear? Why or why not?

6.

List two reasons for not propping inoculating loops and needles in the Bacticinerator during sterilization.

7.

How long does it take to sterilize an inoculating loop or needle?

8.

When removing a lid from a lid or a bottle using aseptic technique, what do you do with the lid?
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Staining
Introduction Bacteria have almost the same refractive index as water. This means when you try to view them using a microscope they appear as faint, gray shapes and are difficult to see. Staining cells makes them easier to see. Simple stains use only one dye that stains the cell wall of bacteria much like dying eggs at Easter. Differential stains use two or more stains and categorize cells into groups. Both staining techniques allow the detection of cell morphology, or shape, but the differential stain provides additional information concerning the cell. The most common differential stain used in microbiology is the Gram stain. Bacteria have three basic shapes or morphological types. Round cells are known as cocci, rod-shaped cells are bacilli, and spiral-shaped cells are spirilla.

Cocci

Bacilli

Spirilla

Principle Simple Stain: The simple stain consists of one dye. The dye adheres to the cell wall and colors the cell making it easier to see. Gram Stain: The Gram stain is a differential stain. Four different reagents are used and the results are based on differences in the cell wall of bacteria. Some bacteria have relatively thick cell walls composed primarily of a carbohydrate known as peptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycan and lipopolysaccharides. Peptidoglycan is not soluble in non polar or organic solvents such as alcohol or acetone, but lipopolysaccharides are nonpolar and will dissolve in nonpolar organic solvents.

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Crystal violet acts as the primary stain. This stain can also be used as a simple stain because it colors the cell wall of any bacteria. Grams iodine acts as a mordant. This reagent reacts with the crystal violet to make a large crystal that is not easily washed out of the cell. At this point in the staining process all cells will be the same color. The difference in the cell wall structure is displayed by the use of the decolorizer. A solution of acetone and alcohol is used on the cells. The decolorizer does not affect those cell walls composed primarily of peptidoglycan but those with the lipid component will have large holes develop in the cell wall where the lipid is dissolved away by the acetone and alcohol. These large holes will allow the crystal violet-iodine complex to be washed out of the cell leaving the cell colorless. A counterstain, safranin, is applied to the cells which will dye the colorless cells. The cells that retain the primary stain will appear blue or purple and are known as Gram positive. Cells that stain with the counterstain will appear pink or red and are known as Gram negative. The lipopolysaccharide of the Gram negative cell not only accounts for the staining reaction of the cell but also acts as an endotoxin. This endotoxin is released when the cell dies and is responsible for the fever and general feeling of malaise that accompanies a Gram negative infection. When reporting a Gram stain you must indicate the stain used, the reaction, and the morphology of the cell. Round, purple (blue) cells would be reported as Gram positive cocci and rod-shaped, purple (blue) cells would be reported as Gram positive bacilli. There are standard abbreviations that may be used for these reports. GPC GNC GPB GNB Gram positive cocci Gram negative cocci Gram positive bacilli Gram negative bacilli

The spiral-shaped bacteria of medical importance do not Gram stain well and are usually demonstrated using a dark-field microscope. There are no standard abbreviations for Gram stain reactions for the spirilla. Procedure Simple stain Materials Heat-fixed bacterial smears Methylene blue, Crystal violet, or Safranin to act as simple stain Bibulous paper or paper towels Microscope 1. 2. Cover the label on the slide with tape. Place the slide on the staining rack and flood the slide with stain for 1 minute.
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3.

Rinse the slide with tap water, tilting the slide slightly to rinse all the stain from the slide. Tap the slide gently to remove excess water. Place a piece of bibulous paper or paper towel on the lab table and put the slide on it. Fold the paper over the slide and gently blot the slide to remove the water. Examine the stained smear with the microscope and record your results in the chart below.

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Organism
Staphylococcus aureus E. coli
Gram Stain Materials Heat-fixed bacterial smears Gram stain reagents Crystal violet Grams iodine Acetone-alcohol decolorizer Safranin Bibulous paper or paper towels Microscope 1. 2.

Results

Cover the label on the slide with tape. Place the slide on the staining rack and flood with crystal violet for 1 minute. Rinse the slide with tap water, tilting the slide slightly to rinse all the stain from the slide. With the slide slightly tilted, drop a few drops of Grams iodine on the slide to rinse off the last of the rinse water. Place the slide flat and flood with Grams iodine for 1 minute. Rinse the slide with water as in step 3. With the slide tilted slowly drop acetone-alcohol decolorizer on the slide. Blue color will run from the smear. Continue to apply decolorizer drop-by-drop until the blue stops running from the smear.
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Immediately rinse with water. With the slide slightly tilted add safranin to the slide to replace the rinse water then lay the slide flat and flood the slide with safranin for 30 seconds. Rinse safranin from the slide with tap water. Gently tap the slide to remove excess water. Place a piece of bibulous paper or paper towel on the lab table and put the slide on it. Fold the paper over the slide and gently blot the slide to remove the water. Examine the stained smear with the microscope and record your results in the chart below.

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Organism
Staphylococcus aureus E. coli

Results

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Review Questions What is the purpose of staining bacteria?

List and describe the three basic bacterial shapes.

What are the differences between a simple stain and a differential stain?

What is the most common differential stain used in microbiology?

What is the basis for Gram stain results between different bacteria?

List the reagents used in the Gram stain and tell the function of each.

What would be the proper way to report each of the following if they had been Gram stained? Purple (blue), round cells Pink (red), rod-shaped cell Pink (red), round cells Purple (blue), rod-shaped cells
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Culturing and Isolation Techniques

Introduction Microorganisms must have a constant nutrient supply if they are to survive. Free-living organisms acquire nutrients from the environment and parasitic organisms acquire nutrients from their host. When trying to grow microbes in the lab adequate nutrition must be provided using artificial media. Media may be liquid (broth) or solid (agar). Any desired nutrients may be incorporated into the broth or agar to grow bacteria. Agar is the solidifying material used in solid media. It is an extract of seaweed that melts at 100o C and solidifies at about 42o C. Most pathogenic bacteria prefer to grow at 37o C so agar allows for a solid medium at incubator temperatures. Since agar remains solid until reaching 100o C, thermophiles (heat-lovers) that prefer temperatures above 50o C for growth can still be grown on solid media. Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth. On agar, masses of cells, known as colonies, appear after a period of incubation. Certain techniques will allow bacterial cells to be widely separated on agar so that as the cell divides and produces a visible mass (colony), the colony will be isolated from other colonies. Since the colony came from a single bacterial cell, all cells in the colony should be the same species. Isolated colonies are assumed to be pure cultures. Principle A mixed culture contains two or more bacterial species that are known and can be easily separated based on cultural or biochemical characteristics. Culturing techniques provide a means for maintaining adequate nutrition for the organisms so they can continue to survive. As organisms grow in a culture they consume the available nutrients and periodically need to be transferred to fresh media to continue to grow. Certain culturing techniques not only provide the organisms with a fresh supply of nutrients but also allow for the separation of bacterial cells to obtain isolated colonies. These culturing procedures are known as isolation techniques. Streak plates allow for the growth of isolated colonies on the surface of the agar. An isolated colony is a colony that is not touching any other colonies and is assumed to be a pure culture. These colonies are easily accessible for performing staining and identification procedures. They also show colonial morphology that may be useful in identifying the organism. Part of the identification of any organism includes a description of colonial morphology. Since organisms may grow differently on different media, the type of media used must be included as a part of any colonial morphology. Other elements of a colonial description include colony color, hemolysis (if grown on blood agar), form, elevation and margin.
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Form refers to the overall appearance of the colony. Elevation is the height the colony achieves on the surface of the agar. The appearance of the edge of the colony is referred to as the margin.

FORM

Circular > 1mm Punctiform < 1 mm

Irregular

ELEVATION
________________________________________________________ Flat Convex Umbonate

MARGIN

Entire

Undulate

Curled

The pour plate is used for counting organisms in a solution. A standard volume of solution is mixed in the liquefied agar. Each organism in the solution is separated from all others. When the agar solidifies the cells are trapped in the agar and develop into colonies. Each colony can be counted and represents a single cell in the original solution. If a milliliter of solution is mixed in the agar then the number of colonies represents the number of organisms per milliliter of solution. Usually a portion of a milliliter is mixed in the agar so the number of colonies counted must be multiplied by the dilution factor to determine the number of organisms in a milliliter of solution. When counting colonies in agar it is difficult to accurately count more than 300 colonies on a plate. Less than 30 colonies on a plate are considered statistically insignificant. When evaluating a solution for bacteria a series of dilutions is usually made and cultured. The plate with 30-300 colonies is counted and the number multiplied by the dilution factor for that plate to determine the number of bacteria per milliliter in the original solution. This method is used to evaluate the number of organisms in milk, drinking water, and even the water at the beach. While the cells grow and are isolated from each other in a pour plate, they will not develop typical colonial morphology and are not easily accessible for further testing.
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Procedure Inoculation of a Broth Culture Materials Mixed Culture in broth Inoculating loop Bacticinerator Incubator Sterile nutrient broth Students work individually. 1. 2. 3. Label the sterile nutrient broth with the source of the culture and your initials. Sterilize the loop. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube. Insert the loop into the sterile broth tube and swirl gently. Sterilize the loop. Incubate the broth at 37o C for 24-48 hours. Observe broth for turbidity. Record results in table at end of the procedure section.

4. 5. 6.

Inoculating an Agar Slant Materials Mixed Culture in broth Inoculating loop Bacticinerator Incubator Sterile nutrient agar slant Students work individually. 1. Label the sterile nutrient agar slant with the source of the culture and your initials. Sterilize the loop.

2.

3.

Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube.
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4.

Insert the loop into the sterile agar slant tube and starting at the base of the slant, draw the loop up the slant. Do not penetrate the agar. Sterilize the loop. Incubate the slant at 37o C for 24-48 hours. Observe the slant for growth. Record results in table at end of the procedure section.

5. 6.

Streak Plate Materials Mixed Culture in broth Inoculating loop Bacticinerator Incubator Sterile nutrient agar plate Students work individually. 1. Label the sterile nutrient agar plate with the source of the culture and your initials. Sterilize the loop. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube. Lift the agar plate from the lid and streak about half of the plate. The loop should be parallel to the agar surface to prevent digging into or gouging the agar.

2. 3.

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Return the plate to the lid. Sterilize the loop. Lift the agar plate and make one streak into the inoculated portion of the plate. Finish by streaking about onefourth of the uninoculated plate.

6.

Return the plate to the lid. Sterilize the loop. Lift the agar plate and make one streak into the second inoculated portion of the plate. Finish by streaking the remaining one-fourth of the uninoculated plate. Sterilize the loop.

7.

Place the plate in a 37o C incubator for 24-48 hours. Observe for growth and record your results in the table provided at the end of the procedure section.

29

Pour Plate Materials Mixed Culture in broth Inoculating loop Bacticinerator Incubator Nutrient agar deep, liquefied Sterile petri dish Sterile pipette Students work in pairs. 1. Label the bottom of the sterile petri plate with the source of the culture and your initials. Turn the plate so the lid is facing up. Obtain two tubes of liquefied nutrient agar, one for each student in the pair. The nutrient agar was boiled (100o C) to melt the agar. Agar at that temperature would kill bacteria, so the agar has been cooled to 60o C and held in a water bath to maintain that temperature. This should not kill the bacteria when they are introduced to the liquid agar and will also reduce the amount of condensation that will collect on the lid of the petri dish. Work quickly. The agar will solidify at 42o C. One student of the pair should aseptically transfer two drops of the mixed culture broth to one of the agar tubes. The other student should aseptically transfer one drop of the mixed culture broth the second agar tube. Mix the tubes by rolling the tubes between your hands, then pour the inoculated liquid agar into a labeled sterile petri dish. Gently move the dish in a figure eight to completely cover the bottom of the dish with agar. Allow the agar to solidify. Add to the labeling the amount of mixed culture used in the agar. A milliliter contains approximately 20 drops. Two drops would be approximately 0.1 ml and 1 drop would be approximately 0.05 ml. Incubate the plates at 37o C for 24-48 hours. Examine the plates for growth and record the results in the table below.

2.

3.

4.

5.

Culture Broth Slant Streak Plate Pour Plate 0.1 ml Pour Plate 0.05 ml

Growth

Isolation

30

Review Questions How can you tell growth has occurred in a broth culture?

What is the purpose of agar?

At what temperature does agar liquefy?

At what temperature does agar solidify? Why is liquefied agar cooled to 60o C before adding organisms?

List two methods for obtaining isolated colonies.

What is the primary purpose of the streak plate?

How many colony types did you observe on your streak plate?

Describe each colony type observed using standard terms.

What is the primary purpose of the pour plate?

31

How many colonies did you observe on the 0.1 ml pour plate?

How many colonies did you observe on the 0.05 ml pour plate?

If possible determine the number of organisms in the original mixed culture broth. The dilution factor for 0.1 ml is 10 and for 0.05 ml the dilution factor is 20.

What is the general formula for determining the number of organisms in a solution using a pour plate?

Serial dilutions are made of milk. The following information is collected. Dilution 1:100 1:1000 1:10000 Number of Colonies 312 262 22

How many organisms are in a milliliter of the milk tested?

32

Identification of Gram Positive Cocci Staphylococcus

Introduction The genus Staphylococcus contains both pathogenic and non-pathogenic organisms. They do not produce endospores but are highly resistant to drying, especially when associated with organic matter such as blood, pus, and other tissue fluids. Most staphylococci are found routinely on the surface of the skin. Breaks in skin and mucous membranes allow entrance of these organisms into the body where they may cause disease. The three major species include Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The latter two are rarely implicated in disease, but have been isolated in cases of endocarditis and urinary tract infections under certain circumstances. Staph. aureus is considered the pathogenic strain, causing abscesses, boils, carbuncles, acne and impetigo. Less commonly, pneumonia, osteomyelitis, endocarditis, cystitis, pyelonephritis, and food poisoning have been attributed to this organism. These three strains of staphylococci can be distinguished from each other by a number of biochemical tests. Principle The identification of organisms is based on cellular, cultural and biochemical characteristics. All species of Staphylococcus are Gram positive cocci. On nutrient agar they tend to be white, circular, entire, convex colonies. On blood agar Staphylococcus aureus may show hemolysis of the agar in the area around the colony. Additional biochemical tests that are useful in separating the Staphylococcus species include catalase, coagulase, growth and fermentation of mannitol salt, and resistance or susceptibility to the antibiotic novobiocin. The catalase test determines if the organism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. 2 H2O2
__catalase__

> 2 H2 O + O2

This enzyme allows organisms to breakdown harmful metabolites of aerobic respiration and may be seen in aerobic and facultatively anaerobic organisms. There are other enzymes that some organisms produce to handle toxic endproducts of metabolism so not all aerobes or facultative anaerobes produce catalase. Pathogenic organisms require mechanisms to help them overcome host defense mechanisms. One mechanism involves coating the bacterial cells in a body substance, such as fibrin, to fool the immune system. The coating of a natural body substance will
33

not trigger an immune response. The enzyme coagulase causes fibrin to be deposited on bacterial cells. Some organisms can not tolerate a high osmotic pressure. Media containing higher than normal salt concentrations may inhibit the growth of these non-tolerant organisms. Mannitol salt agar contains a high salt concentration so only salt tolerant organisms will grow on it. Additionally, mannitol salt agar contains the sugar mannitol. Some organisms can utilize mannitol as a food source and will produce acid endproducts from this metabolism. Since this process is invisible an indicator is added to the media to detect changes in pH. Phenol red is the indicator used in mannitol salt agar. It is red at a neutral pH but turns yellow if conditions in the media become acidic. Antibiotic susceptibility is another test that can be used to identify organisms. A filter paper disc is impregnated with an antibiotic, in this case novobiocin. When the disc is placed on agar, the antibiotic diffuses through the agar. An organism susceptible to the antibiotic will be unable to grow on the media containing the antibiotic. A zone of inhibition (no growth) will be seen around the disc. The size of the zone indicates the resistance or susceptibility of the organism to the antibiotic. Procedure Catalase 1. 2. Place a drop of 3% H2 O2 on a glass slide. Touch a sterile loop to a culture of the organism to be tested and pick up a visible mass of cells. Mix the organism in the drop of hydrogen peroxide. Observe for immediate and vigorous bubbling. Dispose of slide in the contaminated slide container.

3. 4. 5.

Interpretation: Bubbling indicates a positive (+) test and scant or no bubbling indicates a negative (-) test. Coagulase 1. Dispense 1 drop of Test Latex onto one of the circles on the reaction card and 1 drop of Control Latex onto another circle. Touch a sterile loop to a culture of the organism to be tested and pick up a visible mass of cells. Mix the cells in the drop of Test Latex. Repeat Step 2 for the Control Latex.
34

2.

3.

4.

Pick up and hand rock the card for up to 20 seconds and observe for agglutination or clumping of the latex particles. Dispose of the reaction card in the biohazard container.

5.

Interpretation: Agglutination of the Test Latex with no agglutination of the Control Latex is considered a positive (+) test for coagulase. No agglutination in either the Text Latex or Control Latex is considered negative (-) for coagulase. All reactions occurring after 20 seconds should be ignored. If agglutination occurs in the Control Latex the agglutination is due to some factor other than the enzyme coagulase and the test results are invalid. Mannitol Salt Agar 1. 2. Label a tube of mannitol salt agar with the organism to be tested and your initials. Using a sterile loop transfer the organism to be tested to the surface of the mannitol salt agar slant. Incubate the tube at 35o C. for a minimum of 18 hours. Examine the tube for evidence of growth on the slant and for a color change from red to yellow. Remove the markings from the tube using Grams decolorizer on a paper towel and place the tube in the designated area for disposal.

3. 4.

6.

Interpretation: Two different characteristics of the organism are determined with this agar. The first is the organisms ability to tolerate a high salt environment. Evidence of growth on the slant indicates the organism can grow in a high salt environment. Organisms that can ferment the sugar mannitol produce an acid end product that changes the red pH indicator in the media to yellow. Any yellow in the media is considered a positive test for mannitol fermentation. It is possible for organisms to grow on the media and not ferment mannitol. Novobiocin Susceptibility 1. 2. 3. Divide a nutrient agar plate into three sections. Label a section with the name of the organism to be tested. Using a sterile loop transfer the test organism to the plate and streak the section for confluent growth. Aseptically transfer a novobiocin antibiotic disc to the center of each streaked area. Gently press the disc to the surface of the agar.
35

4.

5. 6. 7.

Invert the plate and place in the incubator for a minimum of 18 hours. Examine the plate for a zone of inhibition of growth around the antibiotic disc. Using a metric ruler, measure the diameter of the zone of i nhibition and record the measurement in millimeters (mm). Discard the plate in the biohazard container.

8.

Interpretation: A zone of growth inhibition 17 mm or less in diameter indicates resistance (R) to novobiocin. If the zone is greater than 17 mm the organism is susceptible (S) to novobiocin.

LABORATORY INSTRUCTIONS
Cultures provided: Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Students work individually unless otherwise noted. 1. Make a smear of one of the organisms provided. (See page 18) Complete the remainder of the laboratory work before heat fixing, staining and examining the smear. Perform a catalase test on all organisms. Select one of the three organisms and perform a coagulase test. Allow the other members of your group to observe your results. Observe the results of the other 2 organisms. Select one of the three organisms and inoculate a mannitol salt agar slant. As in step 3, observe the results of all three organisms Test each organism for novobiocin susceptibility. Each person should test all three organisms. Record all results on the Laboratory Record Sheet. (Page 37) As time permits, Gram stain the smear prepared in Step 1 (Page 22).

2. 3.

4.

5.

6. 7.

36

MCB 1000L Identification of Staphylococcus Test Gram Stain Catalase Coagulase Growth on mannitol salt Mannitol fermentation Novobiocin susceptibility All species of Staphylococcus are Gram ___________________ ____________________ and positive for the ___________________________ test. Also, all Staphylococcus species tolerate ___________________________ as indicated by their growth on mannitol salt agar. Which test differentiates Staph. aureus from the other species of Staphylococcus? Staph. aureus Staph. epidermidis Staph. saprophyticus

How can you differentiate Staph. epidermidis from Staph. saprophyticus?

37

Identification of Gram Positive Cocci Streptococcus

Introduction Members of the genus Streptococcus are responsible for disease as well as being part of the normal flora of humans. Among the diseases caused are bacterial pneumonia, meningitis, tonsillitis, endocarditis, scarlet fever, erysipelas, and urinary tract infections. Streptococcus species are also found normally in the mouth and on the skin surface. The streptococci are classified by two major methods: hemolytic activity and serologic classification of Lancefield.

Classification Based on Hemolytic Activity

When grown on sheep blood agar, streptococci display one of three types of hemolysis of the red blood cells in the agar. Alpha hemolysis --The red blood cells in the media are partially digested producing a greening of the agar. Beta hemolysis--The red blood cells in the media are completely digested producing a clearing of the agar. Gamma hemolysis--No change is noted in the agar. The red blood cells are not affected by the organism.

Expected Hemolysis Streptococcus pyogenes

alpha
never never

beta
always usually sometimes never sometimes

gamma
never sometimes usually never usually

Streptococcus agalactiae
sometimes

Streptococcus bovis
always

Streptococcus pneumoniae
sometimes

Enterococcus faecalis

38

Classification Based on Lancefield Proteins

Rebecca Lancefield, working with various streptococcal species, discovered proteins in the cell wall that were unique to certain organisms. These proteins were labeled Group A, Group B, Group C, and so on through Group M. Currently three Lancefield Groups are of medical importance: Group A, Group B, a nd Group D. Of the organisms used in this lab the following correlations apply: Group A Strep--Streptococcus pyogenes Group B Strep--Streptococcus agalactiae Group D Strep--Streptococcus bovis, Enterococcus (Streptococcus) faecalis Streptococcus pneumoniae does not possess Lancefield proteins and is not classified in one of the Lancefield groups. Viridans streptococci is the term applied to alpha hemolytic Streptococcus species that lack Lancefield proteins. Principle All Streptococcus species are Gram positive cocci. Some will only grow on an enriched agar, such as 5% sheep blood agar. On sheep blood agar the colonies are usually gray, punctiform, convex, and entire. Various species display alpha, beta or gamma hemolysis. Important biochemical tests include catalase, bacitracin susceptibility, optochin susceptibility, growth in high salt broth, hemolysis patterns seen with the CAMP test, and the ability to hydrolyze esculin. The bacitracin and optochin susceptibility tests are similar to the no vobiocin susceptibility test used for the identification of Staphylococcus species. Filter paper discs impregnated with the appropriate chemical are placed on an agar surface. The chemical diffuses through the agar. Organisms that are susceptible to the chemical will not grow on the agar containing the chemical. The size of the zone of growth inhibition determines the organisms susceptibility to the chemical. CAMP factor is a diffusable protein produced by certain species of Streptococcus. This factor will react with the beta toxin produces by Staphylococcus aureus to rapidly lyse sheep red blood cells. When a CAMP producing Streptococcus is grown near a beta toxin producing strain of Staphylococcus aureus a definite hemolytic pattern is produced. Only a few organisms can tolerate a salt concentration of 6.5% NaCl. Those that can will grow in high salt broth. Bile esculin agar contains bile that inhibits the growth of many organisms. Some organisms can hydrolyze esculin to esculetin and dextrose. Esculetin will react with ferric citrate in the media to produce a black-brown product.
39

Procedures Bacitracin Susceptibility 1. Divide a sheep blood agar plate into four quadrants. 2. 3. Label a quadrant with the name of the organism to be tested. Using a sterile loop aseptically transfer the test organism to the plate and streak the quadrant for confluent growth. Aseptically transfer a bacitracin disc (A disc) to the center of the quadrant. Forceps may be used to position the disc. Gently press the disc to the surface of the agar but do not embed the disc in the agar. Invert the plate and place in the incubator for a minimum of 18 hours. Examine the plate for a zone of inhibition of growth around the disc. When finished discard the plate in the biohazard container.

4.

5. 6.

Interpretation: Any zone of inhibition of growth is considered positive (+) for this test. If a red ring can be seen around the disc this is considered a positive test. This test should be done only on organisms that display beta hemolysis . Optochin Susceptibility 1. 2. 3. Divide a sheep blood agar plate into four quadrants. Label a quadrant with the name of the organism to be tested. Using a sterile loop aseptically transfer the test organism to the plate and streak the quadrant for confluent growth. Aseptically transfer an optochin disc (P disc) to the center of the quadrant. Forceps may be used to position the disc. Gently press the disc to the surface of the agar but do not embed the disc in the agar. Invert the plate and place in the incubator for a minimum of 18 hours. Examine the plate for a zone of inhibition of growth around the disc. Using a metric ruler, measure the diameter of the zone of inhibition and record the measurement in millimeters (mm). When finished discard the plate in the biohazard container.

4.

5. 6.

Interpretation: A growth inhibition zone of 15-30 mm is considered a positive (+) test. Zone sizes of less than 15 mm are considered negative (-) for this test. This test should be done only on organisms that display alpha hemolysis.
40

CAMP Test 1. Obtain a sheep blood agar plate that has been prepared for a CAMP test by having Staphylococcus aureus streaked in a single line down the center of the plate. Lines have been drawn on the plate perpendicular to the Staph. streak. These will act as guidelines for inoculating the plate. Label one of the lines on the CAMP plate with the organism to be tested. Using a sterile loop obtain a sample of the test organism. Using a single streak and moving from the outer edge of the CAMP plate toward the Staph. steak, inoculate the CAMP plate with the test organism. Do not allow the test organism to directly touch the Staph. streak or streak across the Staph. streak. The test organism should be streaked using one of the perpendicular lines as a guide. Invert the plate and place it in the incubator for a minimum of 18 hours. Observe the plate for the development of a distinct arrowhead pattern of hemolysis where the test organism and the Staph. almost touch. Discard the plate in the biohazard container.

2.

3.

4. 5.

6.

Interpretation: The arrowhead hemolysis pattern is considered positive (+) for this test. No hemolysis or indistinct hemolysis patterns are considered negative (-) for this test. This test should be done only on organisms that display beta or gamma hemolysis . Bile Esculin 1. 2. Label a bile esculin slant with the organism to be tested and your initials. Using a sterile loop transfer the organism to be tested to the surface of the bile esculin slant. Incubate the tube for a minimum of 18 hours. Examine the tube for a definite blackening of the agar. Remove the markings from the tube using Grams decolorizer on a paper towel and place the tube in the designated area for disposal.

3. 4. 5.

Interpretation: Blackening of the agar is considered positive (+) for this test. No change in the color of the agar is considered negative (-) for this test. This test should be done on all suspected streptococci.
41

High Salt 1. 2. 3. 4. Label a high salt broth tube with the organism to be tested and your initials. Using a sterile loop transfer the organism to be tested to the broth. Incubate the tube for a minimum of 18 hours. Examine the tube for evidence of growth (turbidity). It may be helpful to compare the tube to an uninoculated tube. Do not agitate the tubes before you examine them. Remove the markings from the tube using Grams decolorizer on a paper towel and place the tube in the designated area for disposal.

5.

Interpretation: Organisms that can tolerate a high salt environment (6.5% NaCl) will grow in this broth causing the broth to become cloudy or turbid. Turbidity is considered positive (+) for this test. Organisms that can not tolerate the high salt environment will not grow and the broth will remain clear. Clear broth is considered negative (-) for this test. This test should be done on all suspected streptococci.

LABORATORY INSTRUCTIONS Cultures provided: Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Enterococcus (Streptococcus) faecalis Streptococcus bovis Students work individually unless otherwise noted. 1. Make a smear of one of the organisms provided (See page 18). Complete the remainder of the laboratory work before heat fixing, staining and examining the smear. Perform a catalase test (Page 34) on all organisms and record your results on the Laboratory Worksheet (Page 44). Examine all cultures for hemolysis and record your observations on the Laboratory Worksheet (Page 44). Refer to your Laboratory Worksheet (Page 44) and on all beta hemolytic organisms set up a Bacitracin Susceptibility test.

2.

3.

4.

42

5.

Refer to your Laboratory Worksheet (Page 44) and on all alpha hemolytic organisms set up an Optochin Susceptibility test. Refer to your Laboratory Worksheet (Page 44) and on all beta and gamma hemolytic organisms set up a CAMP Test. Work in lab groups to get all required organisms tested, but be sure each member of the group sets up one test. Organisms may be used more than once in your group if necessary. Working in groups, set up a bile esculin slant on all organisms. Each member of the group must set up at least one test. Working in groups, set up a high salt broth on all organisms. Each member of the group must set up at least one test. After appropriate incubation, examine all tests and record results on the Laboratory Worksheet (Page 44). As time permits, Gram stain the smear prepared in Step 1 (Page 22).

6.

7.

8.

9.

10.

43

MCB 1000L Identification of Streptococcus Test* Gram Stain Catalase Hemolysis Bacitracin Optochin CAMP Test Bile Esculin High Salt *If a test is not done on an organism because it is an inappropriate test for that organism, mark the results box with a large X. What characteristic do Staphylococcus and Streptococcus share? Strep. pyogenes Strep. agalactiae Strep. pneumoniae Enterococcus faecalis Strep. bovis

What test would distinguish Staphylococcus from Streptococcus?

An organism is GPC, catalase negative, and alpha hemolytic. List all appropriate tests for identification of this organism.

An organism is GPC, catalase negative, and beta hemolytic. List all appropriate tests for identification of this organism.

An organism is GPC, catalase negative, and gamma hemolytic. List all appropriate tests for identification of this organism.

Once you know an organism is GPC, what test should you do next?

44

Throat Culture
Introduction The human mouth has numerous and varied organisms as part of its normal flora. Both aerobes and anaerobes flourish is this warm, moist environment. Virtually every type of microorganism can be found in the mouth. The most prevalent are the viridans streptococci. These alpha hemolytic organisms account for most of the organisms that grow aerobically in a throat culture. In addition to these Gram positive cocci, numerous species of Staphylococcus may also be found. Neisseria, Branhamella and the anaerobic Veillonella comprise the majority of the Gram negative cocci found in the mouth. Various Gram negative bacilli, such as Haemophilus species and Klebsiella pneumoniae, are also present. The nonpathogenic Corynebacterium, or diphtheroids, are also alpha hemolytic. Diphtheroids are pleomorphic Gram positive bacilli. Spirochetes, a few yeasts and occasional protozoa round out the normal mouth flora. These organisms are commensals that probably protect us from other organisms that may enter our mouths. The presence of our normal flora prevents other organisms from finding space or nutrients to support their growth. While our normal flora potentially protects us from certain diseases, they do contribute to one. The organisms of the mouth contribute to the development of dental caries. Certain organisms adhere to the teeth forming a network for others to adhere. These organisms produce the plaque found on your teeth. Some of the organisms involved in plaque metabolize sugars found in the mouth to acids that etch the tooth enamel and weaken it. If the tooth enamel is damaged, organisms can penetrate to the pulp of the tooth damaging it. Regular removal of these organisms and plaque helps prevent tooth decay. Principal The one organism responsible for disease in the throat is Streptococcus pyogenes or Group A Strep. This organism is beta hemolytic and not part of the normal throat flora. Sheep blood agar provides the enrichment necessary for growing many of the Streptococcus species and also acts as a differential media. The hemolysis produced on sheep blood agar he lps separate the normal alpha hemolytic organisms from the pathogenic, beta-hemolytic Streptococcus pyogenes. Organisms that grow in the throat also need special atmospheric conditions to grow. These organisms are exposed to the higher carbon dioxide content in exhaled breath. To successfully grow these organisms this carbon dioxide rich atmosphere must be reproduced. Organisms that require less oxygen are known as micoraerophiles. In the laboratory this atmosphere may be produced by placing the plates in a large jar, lighting a candle in the jar and

45

replacing the lid. As the candle burns, some of the oxygen in the jar is converted to carbon dioxide. Typically pharyngitis would cause redness and possibly pockets of pus on the back of the throat. When culturing a throat these areas indicating inflammation should be swabbed to provide the specimen. Usually a swab in a protective plastic sleeve (Culturette) is used to take a throat culture. Once the specimen has been taken the swab is returned to its protective sleeve and an ampule of transport media is broken in the bottom of the sleeve. Transport media is a special purpose media that contains balanced salts to protect the specimen from pH changes and keeps the swab moist while in transit to the laboratory for culturing. Nutrients are not provided so growth does not occur but the organisms can survive for several hours in the transport media, particularly if refrigerated. Procedure 1. 2. 3. Obtain a sheep blood agar plate, sterile swab and tongue depressor. Label the agar plate with your patients name. Using the tongue depressor, flatten the patients tongue. Having the patient say Ahhhh helps flatten the tongue. Being careful not to touch any other parts of the mouth, use the sterile swab to firmly swab the back of the patients throat. Use care. Some people have a very strong gag response and this may induce vomiting. Gently roll the swab across the surface of the blood agar plate. Using a sterile loop, streak the plate for isolation. First streak through the area where you rolled the swab and cover approximately half of the plate. Sterilize the loop and streak one quarter of the plate streaking into the original streaking only once. Repeat the procedure for the remaining quarter of the plate streaking into the second streaking only once. Discard the swab and tongue depressor in the biohazard container. Place the plate in a candle jar. The jar will be incubated at 35-37o C for a minimum of 18 hours. Following incubation, examine the plate for the presence of beta hemolytic colonies. A predominance of beta hemolytic colonies would indicate a possible throat infection with Streptococcus pyogenes. When finished examining the plate, discard in the biohazard container.

4.

5.

6.

7.

46

Review Questions 1. List 3 organisms that are considered normal throat flora.

2.

Why is sheep blood agar used for throat cultures?

3.

What organism is pathogenic in the throat?

4.

What incubation conditions are required for throat cultures?

5.

What is the purpose of the candle jar?

6.

Define microaerophile.

7.

What are viridans streptococci?

8.

What are the most predominant aerobic organisms in the throat?

9.

Complete the table below.

Colony Colony 1 Colony 2

Colonial Morphology

Gram Stain Results

47

Identification of Gram Negative BacilliOxidase

Introduction The oxidase test can be used in the identification of GNB to distinguish non-fermenters (oxidase positive) from fermenters (oxidase negative).
Principle

The oxidase test checks for the presence of the enzyme indophenol oxidase. Tetramethyl-para-phenylenediamine (oxidase reagent) will be oxidized in the presence of atmospheric oxygen by indophenol oxidase causing the formation of a dark-purple compound known as indophenol. Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. 2. 3. Students work in groups to complete this lab. Obtain a sterile swab. Touch the swab to the organism being tested. Place one drop of oxidase reagent on the organism on the swab. Using more than one drop of reagent may dilute the color reaction and result in a false negative. Observe the swab for 10-30 seconds for the development of a dark-purple color around the edge of the organism. This is interpreted as a positive test. No color change or a color change after 30 seconds is interpreted as a negative test. Share your results with the other members of your group. Record all results in the chart below. Dispose of the swabs in the biohazard container. The reagent droppers may be discarded is the regular trash can.

4.

5. 6. 7.

Results Oxidase

Pseudomonas aeruginosa

E. coli

Proteus vulgaris

48

Review Questions 1. Based on your results, which organism(s) could be classified as a nonfermenter?

2.

E. coli and Proteus vulgaris are members of the family Enterobacte riaceae so their reactions are representative of the entire family (all members of the family behave the same way). Klebsiella pneumonia is also a member of the family Enterobacteriaceae. What would its oxidase test result be? Is it a fermenter or a non-fermenter?

49

Identification of Gram Negative BacilliUrea

Introduction The urea test can be used in the identification of GNB, particularly those in the family Enterobacteriaceae. Principle If an organism produces the enzyme urease it will break down urea to ammonia and carbon dioxide. urea
urease

> ammonia + carbon dioxide

Ammonia will increase the pH of the media to 8.0 or higher. The media contains phenol red as a pH indicator. At a pH 8.0 or higher the indicator is a bright pink color. If urea is split to ammonia and carbon dioxide the pH change will cause the media to turn bright pink and the test will be considered positive for urease. Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. 2. Work in groups to complete this lab. Urea media may be either a broth or a slant. Obtain urea media and inoculate tubes with the three organisms listed above. Use appropriate aseptic technique when inoculating the tubes. Incubate the tubes for a minimum of 18 hours at 35-37o C. Examine the tubes for a color change. Tubes that are bright pink are considered positive for the test. Any other color change is considered negative. Remove labels from the tubes and discard in the designated area. Record all results in the table below.

3. 4.

5.

Result Urea

Pseudomonas aeruginosa

E. coli

Proteus vulgaris

50

Review questions 1. What enzyme is produced by organisms that can split urea?

2.

What is the indicator used in urea media?

3.

Why does the media turn pink when the test is positive?

51

Identification of Gram Negative BacilliTSI

Introduction The TSI (Triple Sugar Iron) agar provides information concerning glucose fermentation, utilization of the sugars lactose and sucrose, and the anaerobic respiratory process that uses sulfur as the final electron acceptor to produce hydrogen sulfide. This information is useful in the identification of Gram negative bacilli. Principle TSI Agar contains three sugars: glucose (0.1%), lactose (1.0%), and sucrose (1.0%). It also contains phenol red to indicate a change in pH and ferrous sulfate to demonstrate H2S production. Sugar Fermentation Fermentation is an anaerobic process. When sugar is fermented an acid endproduct is produced and sometimes gas. Phenol red turns yellow under acid conditions and red under alkaline conditions. Yellow agaracid productionsugar fermentation The enzymes for glucose fermentation are constitutive enzymes so glucose is the first choice of an organism for fermentation. The acid produced will turn the agar in the tube yellow. Some organisms also produce gas from glucose fermentation. This gas may be trapped in the agar pushing the agar up in the tube or causing cracks or bubbles in the tube. It is possible for the gas to escape around the agar and not be detected. If only glucose can be used the organism quickly uses the available glucose in the tube. In order to survive the organism begins using the protein in the agar as a carbon source. The first step of protein utilization is deamination (forming ammoniaalkaline). Deamination is an aerobic process and only occurs on the slant. Those organisms that can ferment only glucose deaminate proteins to continue to survive and the slant reverts to red due to the alkaline conditions produced. Organisms that can use either sucrose or lactose (or both) will begin to ferment these sugars once the glucose has been consumed. The enzymes required for utilization of these sugars are inducible so the presence of sucrose and lactose in the media will activate the necessary operons. Acid will continue to be produced as a result of the metabolism of the lactose and/or sucrose and the tube will remain yellow. There is a sufficient quantity of either sugar in the media to support the organism for at least 2-3 days.

52

Hydrogen Sulfide (H 2S) Production Anaerobic respiration does not require oxygen since an inorganic salt acts as the final electron acceptor instead of oxygen. Sulfur is one of the anaerobic electron acceptors used by some facultative and obligate anaerobes. Sulfur is readily available in the environment and in media in both organic (amino acids) and inorganic (sulfates) molecules. Hydrogen sulfide is a colorless, volatile liquid. In order to detect its presence in the media, ferrous sulfate is used as an indicator. H2S + ferrous sulfate ferrous sulfide (Black precipitate) H2S production is an anaerobic process so the black precipitate will appear only in the butt of the TSI tube. Reporting Results and Interpretation Black buttH2S + No black in buttH2S Report slant/butt Report A/A A/AG K/A K/AG H2S + H2S -K/K K/H2S Interpretation Glucose and sucrose and/or lactose fermented Glucose fermented with gas production, sucrose and/or lactose fermented Glucose only fermented Glucose only fermented with gas produced Hydrogen sulfide produced Hydrogen sulfide negative No sugars fermented Lactose and sucrose not fermented H2/S production (black butt) is all that can be seen Lactose and/or sucrose fermented H2/S production (black butt) is all that can be seen Yellow agaracidA Red agaralkalineK Gas producedG

A/ H2S

53

Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. 2. 3. Students work in groups to complete this exercise Label a TSI slant with one of the organisms to be tested. All tests in this media rely on anaerobic conditions. To provide this the organism must be introduced into the media, not on the surface. Using a sterile inoculating needle , touch the organism to be tested and stab the TSI media penetrating to the bottom of the tube. When removing the needle, streak the slant. Incubate all tubes for at least 18 hours at 35-37o C. After incubation examine the tubes for color changes. Record all results in the table below. When finished examining the tubes, remove all labels and markings and place in the designated contaminated area.

4. 5.

6.

Organism Pseudomonas aeruginosa E. coli Proteus vulgaris

Fermentation

H2S Production

54

Review Questions Why is the media stabbed when inoculating it?

Why does the slant turn red if only glucose is fermented?

Which organism(s) produced gas? How could you tell?

Which organism(s) produced H2S? How could you tell?

What is the interpretation of the results for Pseudomonas aeruginosa?

What is the interpretation of the results for E. coli?

What is the interpretation of the results for Proteus vulgaris?

Do the TSI fermentation results match the oxidase results?

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Identification of Gram Negative BacilliMotility

Introduction Procaryotic cells (bacteria) have a single strand of protein for a flagellum. The flagellum is the only organelle for motility in the procaryotic cell. Eucaryotic cells may move by using flagella, cilia, or pseudopods. Motility in bacteria indicates that the organism has flagella. Principle Bacterial flagella may be stained using a special flagellar stain to demonstrate their presence. This procedure is somewhat tedious. An alternate way to show bacteria have flagella is to demonstrate their ability to move. Since the only organelle for motility in bacteria is the flagellum, movement indicates the presence of flagella. Media that contains half the agar content as usual is semisolid. It does not pour but is too soft to produce a slant. This consistency will allow bacteria to swim through the agar from the initial inoculation point if they posses flagella. Cells will be distributed along the migration route and will cause the media to become cloudy so the trail will be visible. If a tetrazolium salt (triphenyltetrazolium chloride or TTC) is added to the medium the bacterial presence in the media will be much easier to see. TTC is colorless and soluble in the oxidized form but becomes insoluble and turns red when reduced. Since any metabolic process involves the oxidation of molecules to produce energy, the dye is readily reduced by microbial growth and other microbial activities, such as motility. When TTC is present in the media the cloudy trail left by bacteria swimming through the media is red. The semi-solid agar technique is the most common test for motility in the clinical laboratory. Motility may also be demonstrated using the hanging drop method. A drop containing bacteria is suspended from a cover slip using a depression slide. The slide is then examined to see if the organisms are moving directionally for a distance of 2-3 cell lengths. This is considered true motility. Due to the size of the bacterial cell it is possible to see Brownian movement if the cells are nonmotile. This movement is the result of molecular bombardment of the cells and is not due to the presence of flagella.

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Procedure Organisms used E. coli Klebsiella pneumoniae Enterobacter cloacae 1. 2. Students work in groups to complete this exercise. Obtain a tube of motility media. Using a sterile inoculating needle inoculate the motility media by stabbing halfway into the agar. Incubate for a minimum of 18 hours at 35o C. After incubation, examine the initial stab line. If the line is sharp the organisms did not move and there is no motility. If the line is fuzzy, shows a cloud of growth around it, or is indistinct in any way the organism was able to move away from the initial stab line and is motile. Record all results in the table below. Remove all marks from the tube and discard in the designated area. OPTIONAL: If available, observe the demonstration of the hanging drop technique.

3. 4.

5.

6.

Organism Results
Review Questions

E. coli

Klebsiella pneumoniae

Enterobacter cloacae

What is Brownian movement? Is it motility?

What organelle(s) for motility do bacteria posses?

List three methods that may be used to demonstrate flagella in bacteria.

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Identification of Gram Negative BacilliIMViC

Introduction IMViC is a mnemonic to remember the four biochemical tests being used: Indole, Methyl red, Voges-Proskauer, and Citrate. These four tests help divide the Enterobacteriaceae into two major groupsthe E. coli group and the Enterobacter-Klebsiella group. Principle Indole Organisms that posses the enzyme tryptophanase can break down the amino acid tryptophan to indole. When indole reacts with para-dimethylaminobenzaldehye (Kovacs reagent) a pink -colored complex is produced. Tryptophan is plentiful in most media, but growth on blood agar or chocolate agar produces the best effects. Methyl Red Some organisms produce acid from the metabolism of glucose in a sufficient quantity to produce a pH of 4.4 in the media. These acids are not further metabolized and are said to be stable acids. At a pH of 4.4 or less the pH indicator methyl red is a bright cherry red. Voges-Proskauer Some organisms initially produce acid from glucose metabolism but further metabolize the acid produced to neutral end products, such as acetoin, and 2,3-butanediol. Initially the pH may drop to 4.4 but the neutral end products raise the pH so the methyl red test will be negative. Acetoin and 2,3 -butanediol under alkaline conditions will react with alpha-naphthol (1-naphthol) to produce a mahogany red color. Citrate Citrate contains carbon. If an organism can use citrate as its only source of carbon the citrate in the media will be metabolized. Bromthymol blue is incorporated into the media as an indicator. Under alkaline conditions this indicator turns from green to blue. The utilization of citrate in the media releases alkaline bicarbonate ions that cause the media pH to increase above 7.4.

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Procedure Organisms used E. coli Klebsiella pneumoniae Enterobacter cloacae 1. 2. Students work in groups to complete this exercise. Obtain a DrySlide indole test card. Using a sterile loop transfer cells from an agar plate or slant to the test area on the card. Observe for the development of a pink color within 30 seconds. Record your results in the table provided (Page 60) and discard the test card in the biohazard container. Obtain an MRVP broth and using aseptic technique inoculate the tube. It is important to inoculate this test heavily. Incubate for at least 24 hours at 35o C. Obtain a citrate slant. Aseptically inoculate the slant and incubate for at least 24 hours at 35o C. After incubation of the MRVP broth obtain a spot plate and a sterile dropper. Observe the MRVP for turbidity. If turbidity is not noted the test results are not reliable. The tube may be re-incubated until growth is evident. Place 3 drops of turbid broth into two of the wells on the spot plate. Methyl Red TestTo one well add 1-2 drops of methyl red reagent. Observe for an immediate cherry red color that indicates a positive test. Orange or yellow is considered negative. Record your results in the table provided (Page 60). Voges-Proskauer TestTo the remaining well add 2 drops of alphanaphthol and 1 drop of potassium hydroxide (KOH). Observe for the development of a mahogany red color. The color development takes 20 minutes or longer. Be extremely careful with the KOH. It is caustic and may cause burns if it gets on your skin. The mahogany red color is considered positive. Record your results in the table provided (Page 60). Remove all marks from the MRVP tube and discard in the designated area. Clean the spot plate by covering the surface with disinfectant. Allow the disinfectant to sit for a few minutes, then rinse with water, wash and dry. Return the spot plates to their original location. Observe the citrate for a change from green to blue. Blue is considered positive for this test. Record your results in the table provided (Page 60). Remove all marks from the tube and discard in the designated area.

3.

4.

5.

6.

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Organism E. coli Enterobacter cloacae Klebsiella pneumoniae

Review Questions

Indole

Methyl Red

VogesProskauer

Citrate

What is the IMViC pattern for the E. coli group?

What is the IMViC pattern for the Enterobacter-Klebsiella group?

What is the difference between a reagent and an indicator?

Complete the following table.

Test Indole Methyl Red VogesProskauer Citrate

Substrate

Reagent

Indicator

Positive Result

Is it possible for an organism to be Methyl red and Voges-Proskauer positive? Explain your answer.

What might happen if the MRVP tests are read too soon?

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Disc Diffusion Susceptibility Methods

Introduction When a filter paper disc impregnated with a chemical is placed on agar the chemical will diffuse from the disc into the agar. This diffusion will place the chemical in the agar only around the disc. The solubility of the chemical and its molecular size will determine the size of the area of chemical infiltration around the disc. If an organism is placed on the agar it will not grow in the area around the disc if it is susceptible to the chemical. This area of no growth around the disc is known as a zone of inhibition. Principle Antiseptics, disinfectants and antibiotics are used in different ways to combat microbial growth. Antiseptics are used on living tissue to remove pathogens. Disinfectants are similar in use but are used on inanimate objects. Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus, that kill or inhibit the growth of other organisms, primarily bacteria. Many antibiotics are chemically altered to reduce toxicity, increase solubility, or give them some other desirable characteristic that they lack in their natural form. Other substances have been developed from plants or dyes and are used like antibiotics. A better term for these substances is antimicrobials, but the term antibiotic is widely used to mean all types of antimicrobial chemotherapy. Many conditions can affect a disc diffusion susceptibility test. When performing these tests certain things are held constant so only the size of the zone of inhibition is variable. Conditions that must be constant from test to test include the agar used, the amount of organism used, the concentration of chemical used, and incubation conditions (time, temperature, and atmosphere). The amount of organism used is standardized using a turbidity standard. This may be a visual approximation using a McFarland standard 0.5 or turbidity may be determined by using a spectrophotometer (optical density of 1.0 at 600 nm). For antibiotic susceptibility testing the antibiotic concentrations are predetermined and commercially available. Each test method has a prescribed media to be used and incubation is to be at 35-37o C in ambient air for 18-24 hours. The disc diffusion method for antibiotic susceptibility testing is the KirbyBauer method. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. There is also a zone of intermediate resistance indicating that some inhibition occurs using this antimicrobial but it may not be sufficient inhibition to eradicate the organism from the body.

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The standardized methods for antiseptic and disinfectant testing are more rigorous and more difficult to reproduce in a student laboratory. Two common tests are the Phenol Coefficient Test (a comparison of the effect of the chemical and phenol on several organisms) and the Use Dilution Test (testing the chemical under actual conditions of use). A disc diffusion test can be used to approximate the Use Dilution Test. The chemical under consideration is used to saturate a filter paper disc. This disc is then used to introduce the chemical to the agar for testing. The actual zo ne sizes have not been standardized as in the Kirby-Bauer method, but a comparison of zone sizes for the same chemical among organisms will provide an approximate effectiveness of the chemical. Procedure Kirby-Bauer Antimicrobial Susceptibility Test Organisms to be tested: Staphylococcus aureus E. coli Procedure 1. Students will work independently in the laboratory exercise. 2. Obtain a plate culture of one of the organisms to be tested. 3. Using a sterile loop, emulsify a colony from the plate in the sterile saline solution. Mix thoroughly making sure that no solid material from the colony is visible. 4. Repeat this procedure until the turbidity of the saline solution matches that of the standard available for your class. 5. Dip the swab into the broth culture of the organism. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab to streak a Mueller-Hinton agar plate or a nutrient agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. End by using the swab to streak the outside diameter of the agar. 6. Allow the plates to dry for about 5 minutes.

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7. Antibiotic disks can be placed on the surface of the agar using a dispenser that dispenses multiple disks at the correct distance apart, or by obtaining individual disks and placing them on the surface of the agar using flame sterilized forceps. a. Dispenser method: 1. Obtain the dispenser containing the correct antibiotic disks for the organism you are using. 2. Place the dispenser over the surface of the plate and using the lever/plunger dispense the disks. 3. Using sterile forceps or a loop, gently press the disks onto the surface of the agar, taking care not to press them into the agar. b. Dispensing individual disks: 1. Obtain 6 of the appropriate individual disk dispensers. 2. Using the levers, dispense the disks at equal distances apart on the surface of the agar. 3. Using flame sterilize forceps or a sterile loop gently press the disks onto the surface of the agar. 4. 6 disks may also be individually placed onto the surface of the agar using sterile forceps. 8. Invert the plates and incubate for 24 hours at 37 C. 9. Using a metric ruler measure the diameter of the zone of inhibition (if present) for each antibiotic used. 10. Compare the measurement obtained from the individual antibiotics to the table of standards to determine if the bacterial species tested is resistant or sensitive to the antibiotic. 11. Use the data you collected and that of the rest of the class to fill in the table below. Discard the plates in the biohazard container.

Antibiotic Staph. aureus E. coli

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Antibiotic Tetracycline Ciprofloxacin Enoxacin Erythromycin Penicillin Staphylococci Oxacillin Staphylococci Tobramycin Ceftriaxone Kanamycin Clindamycin Piperacillin Gram negatives Ampicillin Gram negative enterics Staphylococci

Zone Diameter (mm) Interpretation Chart Resistant Intermediate = 14 15-18 = 15 16-20 = 14 15-17 = 13 14-22 = 28 = 10 = 12 = 13 = 13 = 14 = 17 = 13 = 28 11-12 13-14 14-20 14-17 15-20 18-20 14-16

Susceptible = 19 = 21 = 18 = 23 = 29 = 13 = 15 = 21 = 18 = 21 = 21 = 17 = 29

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Antiseptic/Disinfectant Susceptibility Test Organisms used Staphylococcus aureus E. coli Bacillus cereus Pseudomonas aeruginosa 1. 2. Students work individually on this laboratory exercise. Obtain one of the organisms to be tested, 5 nutrient agar plates, and a sterile swab. Dip the swab into the broth culture of the organism. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab to streak a nutrient agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. End by using the swab to streak the outside diameter of the agar. Repeat this procedure for the remaining plates. Place a disc soaked in an antiseptic or disinfectant in the center of each plate. Be sure to label the plates with the organism and chemical used. Incubate the plates in the standard upside down position until the next lab period. Measure the diameter of the zone of inhibition for each chemical. The class will share data so you can fill in the table provided. Discard the plates in the biohazard container.

3.

4.

5.

6.

7.

Chemical Staph. aureus E. coli Bacillus cereus Ps. aeruginosa

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Review Questions What conditions must be held constant when doing disc diffusion procedures?

Define Antiseptic

Disinfectant

Antibiotic

Zone of inhibition

According to your results, which chemical is the most effective? On what do you base this conclusion?

What are the standard tests used for determining the e ffectiveness of antiseptics and disinfectants?

What is the standard method used for antimicrobial susceptibility testing?

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Guidelines for the Identification of Unknown Organisms

Unknown organisms will be grown on 5% Sheep blood agar or Nutrient agar and will be distributed as streak plates with isolated colonies. Each plate will represent a pure culture. Each student is responsible for doing their own work but may ask other students for opinions, advice, or instructions on what to do. The Lab Instruc tor may provide assistance on all but the last two unknowns. (Unknown 3 and Unknown 4) On any of the unknowns, students are allowed to refer to class notes, texts, and any other source of information they may have developed during the course. An Unknown Report Form will be provided for each unknown. Students may have only one form for each unknown. The form must be filled out neatly and completely using blue or black ink. Spelling must be correct and all test notations must be appropriate. This report should be treated as though it were a part of a patient chart. Erasures, liquid paper, and any obliteration of information recorded on the form are not allowed (loss of points). Should errors occur the student must make a single mark through the error, initial, date, and report the corrected result.

+ fd m/d/y ?
The steps for identifying an unknown are Gram stain Colonial morphology Biochemical testing Only the biochemical tests necessary for identification of the organism are appropriate. Unnecessary tests will result in a deduction of points from the final grade. Tests are to be ordered from the lab instructor using the appropriate form. This form must also be filled out completely. A separate order form is used for each unknown. More than one order form may be used for one unknown. If a test is ordered the results must appear on the report form or a brief explanation as to why the test was not done should be made. Failure to do so will result in a loss of points from the final grade. Gram staining and initial spot testing will give the student a general idea concerning the identity of the unknown. Only tests useful for identifying the suspected organism should be performed. A sufficient number of tests should be performed so that the organism may be identified at the next laboratory session.

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Test results must be reported appropriately. Failure to do so may result in a loss of points from the final grade. Most results can be reported with a + or -. That is sufficient. A detailed description of the results is inappropriate. The identity of the unknown should be written appropriately. The genus name should be capitalized but not the species name. If you are in the habit of printing in all upper case letters, be sure to differentiate the first letter of the genus name as larger than the others. Failure to do so will lead to a loss of points on the final grade. Below is an example of the test request form. The top should be completely filled out and the appropriate tests checked. This will be retained by the Laboratory Instructor and attached to your completed Unknown Report Form. It is considered in grading your unknown.

MCB 1000L Media/Test Request Form Name___________________________________Date___________________Unknown Number________ Requested Media/Test Materials Coagulase_______________ Mannitol Salt____________ Novobiocin_______________ Optochin________________ Bacitracin_______________ CAMP__________________ High Salt________________ Bile Esculin______________ TSI_________________ Urea________________ Indole_______________ MRVP______________ Citrate______________ Motility_____________

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Name____________________________ Date Started_______________________

MCB 1000L Unknown Report Form

Unknown Number______________ Gram Stain Results______________ Colony Morphology (include media used) Date________________ Date________________

Test Performed

Results

Date

Identity of Unknown___________________________________________ Date completed__________________

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References
Howard, Barbara J. (ed.) 1987. Clinical and pathogenic microbiology, 2nd ed. Mosby, Baltimore. Finegold, Sidney M., William J. Martin, and Elvyn G. Scott. 1978. Diagnostic microbiology, 5 th ed. Mosby, Baltimore. BBL TM DrySlideTM Indole. 1998. Technical Insert, Becton Dickinson Microbiology Systems, Sparks, Maryland. BBL Oxidase. 1995. Technical Insert, Becton Dickinson Microbiology Systems, Sparks, Maryland.

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