AL-Quds University Faculty of Medicine Abn-Dies, Jerusalem General Microbiology Laboratory Exercises 1. Microscopy-Use of the Oit Immersion Objective 2. «Preparation of bacteriologic media 3. Environmental plate for the growth of bacteria Effect of disinfectants on the growth of bacteria Effect of heat on the growth of bacteria 4, Chemical effects on bacterial growth - pH and microbial growth - Water activity a 5. Isolation of bactei nd osmotic pressure ria (streak plating) 6. Growth characteristics of bacteria 7. The Gram stain/ ‘The acid fast s 8. Antibiotic sensitivity testing 9. Lactobacillus activity in saliva 10. Enzyme linked immunosorbent assay (ELISA). 11. Laboratory final Prepared by Dina M. Bitar Ph.D. Associate professor of Microbiology rao [0595-336652 ive (pis deals aplll 4d2. cusill — parsap LABORATORY RULES “The safety of everyone in the laboratory depends on the observance of these rules. Most of the organisms studied are pathogenic for humans. + 1. Read completely all pertinent exercises before coming to laboratory. ask questions. Your preparedness is essential, and is required for the success and completion of the exercises within the two hour period, 2. Begin no work until the Instructor arrives. 1, All cultures and specimens must be regarded as potentially dangerous. 2... A, laboratory: coat:mustbe-worn: at:allstimes,:primarily to protect your clothing from contamination with infectious material. If your laboratory coat becomes contaminated, itcan be removed and'sterilized. -- ~ 3.: Rating and-smoking:in the laboratory are’ strictly forbidden. In.the interest of your safety, do not put pencils, fingers, or anything else near your. mouth or eyes while working in the laboratory. 4, An unobstructed working area is essential for safety. Keep nothing on your working, “ space-except the material being used in the immediate procedure. Take special care riot to contaminate your microscope, laboratory manual, or other pérsonal property that will -—he removed from the labs infecti 5, Rigid aseptic technique must be followed at all times. The importance of this cannot be over-emphesized, Aseptic procedures will be a matter of daily concern to you for the rest of your medical career, and should be developed, starting with this course, to the point where they become instinctive. | 6. Clear the bench top of everything at the end of each laboratory and wipe it down with disinfectant and paper towels. Lysol disinfectant will be supplied in squeeze bottles. i 7. Discard all materials in appropriate containers. Discard closed culture plates as directed and as soon as-possible in rectangular disposal pans, discard all tubes in discard test tube racks; never lay tubes down in disposal pans. Ifno racks are found in discard pans - ask Instructor. Place discard tubes in upright position in racks. Discard stained slides in waste baskets; put unstained slides in disposal pans.8, Thoroughly label (your name, date, and organism) on all your cultures or they will be abruptly discarded. 9, Incubate the cultures in the 37°C incubator unless otherwise instructed. Your cultures may be incubated in special environments or refrigerated until the next laboratory a period. Wesh your hands with soap and water every time you leave the laboratory for any reason. To reemphasize Rule 1. Ee You must very carefully study each Exercise before coming to class. Tris unlikely that you oe ~~ willbe able to successfully complete the Exercise in only two hours if you are not thordughly familiar with what you are expected to do, and what you are expected to see. Furthermore; lack of preparation ‘will lead to an unsafe situation for. you and. your classmates. You are working with Class 1 and Class 2 bacteria that are all pathogenic for Se humans. Follow all safety procedures strictly and at all times. Expect a quiz every time you come to the-laboratory covering the previous laboratory exercise or the new exercise. You are not allowed to miss any laboratory session without a formally accepted excuse, Evaluation of the laboratory represents 20% of the Final grade and includes lab performance, quizzes, unknowns and laboratory reports.Exercise I Microscope and the Use of the Oil Immersion Objective Microbiology gives the student an opportunity to become familiar with the use of oil-immersion lens. This is a review of the elementary use of the microscope and especially the oil-immersion Jens and assumes some prior use and knowledge and care of the compound microscope. Substage condenser: This compound lens system located below the specimen platform (stage) generally has an iris diaphragm to regulate the amount of light passing into the microscope, The ‘condenser permits the microscopist to focus a cone of light exactly in the object to be examined. Critical illumination is accomplished by first:foou8ing om the object-uinder examination, then closing the iris diaphragm until:the:light is almost ‘extinguished, and then raising or Jowering the entire substage: condenser. until the. interior-edge of the:leaves of the itis diaphragm are sharply -seen.. Then, ‘the iris is-opened until the entire smictoscope field is just. exposed. (With a 10X eyepieceand.a 97X oilimmersion objective the:diameter of the: specimen: area, the real field, being examined is about 0.15 mm.). When the lower power objective lens is used this process is generally impossible to accomplish, however, it is not as necessary at low magnification. What does present a problem at low magnification is the inability to fill the entire field with light, no matter where the substage condenser is positioned. This problem is eliminated in some microscopes by "swinging in" an accessory Jens located at the bottom of the substage condenser. Inclination joint: The microscope” mist “never be tilted when wet preparations are being examined. Objective lenses: These lenses are mounted on the revolving nosepiece. Each lens in color - coded and usually marked with the numerical aperture (N.A) and the focal length and magnification (X). The oil - immersion lens is red banded, 1,25 N.A., 1.9 mm., 97X. What is numerical aperture? What is its importance? The oil-immersion objective lens is the most highly magnifying objective found on light microscopes. Its poorer light gathering ability is obvious, at least in part, from examination of the face diameter of its outermost lens - i's smaller: Remember that the cone of light coming from the substage condenser should be focused'on the specimen. As the light "leaves" the specimen toward the objective lens itis not collimated and it does not all reach the objective. In fact, even without any specimen, the light is bent. The light is bent leaving the microscope slide because the refractive index of glass is 1.52 and air is 1.0. The resultant angular aperture of light is then larger, and in effect less light reaches the objective with a comoomitant decreased ability to sharply distinguish two objects close together as distinctly separate (resolving power). Immersion oil placed between the specimen and the objective Jens substantially reduces the 4bending of light as its reffactive index is the same as glass, i.e, 1.515. The oil -immersion objective is practically useless unless immersion ofl is used, Never put immersion oil on ny objective but the oil-immersion objective, Course and fine adjustment knot Always focus upwards while looking into the eyepiece. Do not lower the body tube toward the specimen while looking into the eyepiece, unless the specimen is first in "good" focus, and then only use the fine adjustment. The safest use of the oil-immersion objective lens generally requires that the specimen be put in focus first using lower power objectives. i.e, with high dry, and then swinging in the par focal-oil immersion lens and focusing by slight adjustment. ~ “After use, swing out the oil-immersion objective and immediately wipe ~ drying immersion oil from the oil-immersion objective with lens'paper or special cotton guazé squafe, if not available, leave the oi-on the objective. Never use anything else to wipe any, microscope lens. Swing in lower power objective and rack down to the safety stop before storing the microscope.Exercise IE Preparation of bacteriologic media Suitable quality culture media for cultivation of microorganisms is required for the successful isolation of aetiological agents. ‘Types of media Bacteriological media can be broadly sub-divided into four categories. 3 1. Ordinary culture media- A Simple nutritionally defined medium e.g. nutrient broth, nutrient agar. 2. Enriched media~A complex medium which is not completely nutritionally defined. Certain organisms do not grow on ordinary nutrient media. They require growth- promoting ingredients such as blood, glucose, serum, egg, etc. e.g. blood agar, chocolate agar and Loeffler medium. 4 3... Enrichment niedis Enrichment media are liquid media containing chemical constituents which inhibit some normal flora and allow pathogens which may be present in very small number in the specimen, to grow thus enriching them. Isolated colonies of these organisms may be obtained by subculturing onto solid media. An example of enrichment media is selenite broth used for primary isolation of enteric bacteria from stool specimens. 4,, Differential and selective media eg. MacConkey- agar contains crystal yiolet and bile salts which inhibit Gram positive bacteria and select for Gram negative rods, MacConkey agar also contains lactose as substrate and neutral red as an indicator. Bacteria fermenting lactose produce acid and this will change the color of the indicator and thus the colonies will tun red. The red lactose fermenting colonies can be differentiated from the pale non-lactose fermenting colonies. Selective media will selectively permit the growth of pathogens and inhibit the commensals. In addition, it may differentiate the pathogen from commensals that grow by the color and opacity of the colonies e.g. blood tellurite medium for Corynebacterium diphtheriae. Transport media are frequently used to sustain the viability of organisms when a clinical specimen is to be transported to the laboratory. The transport medium prevents the ' ‘outgrowth of contaminants during transit and sustains the’ pathogen. Stuart media is an i example of transport media, : In today's exercise you will have the opportunity to prepare different types of media : (nutrient broth, solid agar ete.) and to become familiar with the process of sterilizing and 3 pouring the media into tubes or plates. Presently, a wide range of culture media are available commercially in the form of dehydrated media. These media are simply |“a of reconstituted by weighing the required quantities and by adding distilled water, as per the manufacturer's instructions. Follow the instructions on the media bottles to prepare 500m of each of the following media: Nutrient broth Nutrient agar ‘MacConkey agar Blood agar Trypticase Soy Ager ‘Tryptone ‘Cystéine Yeast Extract AgarExercise IIL Environmental Plate for Growth of Bacteria This exercise will allow you to investigate growth of microorganisms from different environmental areas; Unwanted microorganisms which can get onto culture media/and grow are termed contaminants, Solid nutrient medium will be used to: grow the bacteria..Colonies of bacteria which form on the plate aftervincubation usually result from the division of a single cell, thus represent 2 pure culture, ‘Work in groups of four students. 7 2 Nutrient agar plates 2 Tryptic soy agar (TSA) plates 2 Stetile swabs 1 Tube containing Sm. sterile water Each student in the group does one of the following: a, Place your fingers on the nutrient agar plate and rub them around. Do not cut into the agar surface, b. Use a sterile swab to wipe your tongue then swab the sorface of a TSA plate. Swab the surface of your teeth and gums end then swab the surface of the TSA plete. 4. Dip a sterile swab in a tube of sterile water, and then rub off the desk top. Swab the surface of the nutrient ager plate with the swab. Tneubate the plates at 37°C for 18-24 hrs. Observe the plates for microbiel growth, Compare your results with those of other groups. Describe the morphology of the colonies.Exercise IV Physical and Chemical Effects on Bacterial Growth I. Effect of temperature on growth Each microorganism grows best at its optimum temperature; this is usually, the temperature of the normal habitat. If the temperature is lowered growth will.be inhibited and-if the temperature is raised way above the optimum the organism: will be killed. Work in.groups of four students, 8 tubes of nutrient broth Cultures: Bacillus subtilis and Richérichia coli 1. Inoculate 4 tubes of nutrient broth with Bacillus subtilis, 2. Inoculate the remaining 4 tubes of nutrient broth with Escherichia coli 3. Incubate 1 tube of each culture at 4°C. = Incubate I tube of each culture at 25°C. . Incubate 1 tube of each culture at 37°C. Incubate 1 tube of each culture at 55°C. 4, Observe the tubes after 24-48 hours for growth and again. after 5-7 days incubation. Note the amount of growth in each tube as O = no growth, += little growth, ++ = good growth, classify each culture as mesophile or psychrophile or thermopile. 10 | | | |“Ta Effect of Disinfectants and Antiseptics Work in groups of four students. Sterile Forceps and aleohol 3 disinfectants or antiseptics 9 sterile filter paper dises © Qauttient agar plates 1 TSA plate I sterile swab 1 tube of nutrient broth (for mouth swab) Cultures: £. colt in broth S. aureus in broth ‘Swab of mouth flora in broth, 1. Tnoculate one nutrient agar plate with E.coli by swabing the surface in three directions. * Dispose of the swab in the disinfectant jar. Using the sterile forceps, dip the sterile filter paper discs in each of the antiseptics, drain extra Quid on the side, and place the disc on the surface of the plate. Mark the plate as to the disinfectant used. Incubate at 37°C, 2. Repeat the above procedure for S, aureus 3. Swab the mouth around the teeth, moisten.the swab in a small amount of nutrient broth and then swab the surface of a TSA plate in three directions, Dispose Of the swab in the disinfectant jar. Using the sterile forceps dip the sterile filter paper discs in each of the antiseptics and continue as in step one, 4. Observe the plates after 18-24hs incubation and measure the diameter of the zone of inhibition eround the filter paper dises. aLabs 3 & 4 Work Sheet Part 1: Environmental plate for the growth of bacteria A Part 2: chemical and physical effects on the growth of bacteria ; Objectives: « To investigate the ubiquity of microorganisms .. To assess the value and the benefit from disinfectants in the microbiology lab. . To study the effects of temperature on bacterial growth . To become; familiar: with the following terms: mesophilic, thermophilic, psychrophilic,.optirmum:temperature; disinfectant, antiseptic. 5. To, become:acquainted with an.important microbiology. lab.technique, this is the aseptic technique. » . Bene Materials: « Four N.A plates . 3 TSA plates . 8 nutrient broth tubes = . Broth culture of, coli, S. aureus, Bacillus subtilis . 3 different disinfectants . Swabs | DYswne Results; use (‘++) to express amount of growth Part I: Typeof | Microbialgrowth. | Colonial morphology Me 1. Finger NA 2. Tongue TSA i 3. Gum TSA | 4 Desktop| NA 12Part 2: A. Bffect of temperature on growth Type of E. coli B. subtilis Media Growth 1 temp. _| 24-48 hrs | 5-7 days | 24—48 hrs | 5—7 days 4. ‘Nutrient Broth 25° ‘Nutrient Broth 3? ‘Nutrient Broth 55° | Nutrient Broth Be B, Effeet of Disinfectants on growth Diameter of the zone of inhibition in mm Bacteria ‘Type of Detol | 70% Alcohol | 95% Alcohol Media E, coli NA S. aureus NA Oral mouth flora |TSADiscussion: . Did you get growth of bacteria on all the plates? What does this mean? Were they of the same morphology? Why? . Which disinfectant was the best according to your results? How was the effect of the same disinfectant on different bacteria? Give explanation for your answer? . In theory 70% alcohol is more efficient than 95% alcohol as an antiseptic or disinfectant, why? Did you get such a result, if not, Why? . What is the difference between low temperature and high temperature effects on bacterial growth? . What was the optimal temperature for each organism? . Which bacteria tolerated high temperature (55°)? Explain your result.1 = Exercise V_ Isolation of Bacteria from "simulated clinical specimens" Streak Plating (Overview of Complex, Selective and Differential Media) The metabolic diversity of microorganisms prevents the use of a relatively simple and universally acceptable procedure for the isolation of pathogens. However, it is obvious that a tactically manageable scheme for the'clinical laboratory is a necessity. By the adoption of selective culture techniques the probability of separating and isolating pathogens from unwanted normal flora can be greatly increased. Selective culture techniques commonly rely on the differential response of pathogens and unwanted organisms to the nutritive and inhibitory components in the medium, the atmosphere, and the incubation temperature. Examples of nutritionally selective media are: MacConkey agar is selective for Gram-negative bacteria. MacConkey's agar contains bile salts and crystal violet. which inhibit unwanted bacteria. The inhibited organisms are generally classified as gram positive, the non inhibited, gram-negative. By virtue of these selective features, MacConkey's agar is useful for the isolation of gram - negative pathogenic enteric bacilli which are capable of utilizing Jactose and are not inhibited by the incorporated levels of bile salts or crystal violet. This ‘medium contains lactose as the only carbohydrate and preferentially stimulates the growth of those organisms able to use lactose as a readily available source of carbon. Thus MacConkey agar is also a differential medium, It differentiates between organisms that can ferment lactose and those that cannot férment laétose. Most pathogenic microorganisms, however, are-nutritignally fastidious and require a variety of growth factors in relatively complex media containing natural substances, ¢.8., blood, yeast, serum, milk meat, carbon dioxide, efc. These media are unfortunately often able to support the luxuriant growth of many unwanted microbes present in the same specimen e.g. Blood ager (Muller Hinton agar plus 5% Sheep red blood cells) Another medium which is a complex medium and is selective is Thayer Martin Medium. Basically it is Chocolate agar (Muller Hinton agar plus 5% lysed Sheep red blood cells) with antibiotics and is used: for the- isolation of Neisseria meningitidis and Neisseria gonorrhoeae, the causative (etiologic) agent of gonorhea . These organisms are nutritionally "fastidious" ond their dietary requirements are met in this medium by the combined use of a polysaccharide such as constarch, meat and milk protein, vitamins, hemoglobinamino acids, NAD, cocarboxylase, and other components. The incorporation of the antibiotics vancomycin, colistin and nystatin serves to selectively inhibit unwanted microorganisms, since most neisscria are rather insensitive to these antibiotics at the concentrations used in the medium. In addition to the presence of nutritionally selective and inhibitory substances some media embody a third principle for the isolation of "wanted microorganisms. These media contain metabolic indicator substances, which may change the colonial appearance oforganisms or their surrounding agar. Organisms which can effect these changes are more easily found, Vogel - Johnson (V -J) medium is an example of this type of differential medium, V-J medium contains, among a variety of substances, mannitol, potassium tellurite, a pH indicator, and a relatively high salt concentration. Organisms which can grow on this formulation and produce acid from the fermentation of mannitol will change the phenol red PH indicator yellow, and, if they also reduce the colorless tellurite to dark, insoluble telluride, the colony will blacken as well. Many pathogenic strains of Staphlococcus aureus.can induce both of these changes. Hence, on V-J medium colonies of these bacteria can be more easily picked out in the presence of organisms which lack one or both of these abilities. Unfortunately, there are other bacteria that can also induce these changes, and therefore, these colonial reactions are only presumptive evidence for S. aureus. Nevertheless, in practice V-J medium is helpful in the isolation and identification of S. aureus although. infrequently.used, V.-J.medium-is.2 good: example of a:differential medium. Vogel- Johnson:-medium.not-only.seleets. bacteria. whigh-can-grow in the presence of relatively high and inhibitory salt:concentrations, but: it also may. differentiate those. that do grow by virtue of their specific metabolic activities, ‘Another selective control: js the. variation: of the atmosphere to-inhibit the growth, of unwanted microorganisms. Generally, however, the selective effect of the atmosphere is inadvertent. When we isolate organisms markedly inhibited by oxygen, the obligate anaerobes, such as Bacteriodes or Clostriditan species, we are assured that the obligate aerobes, such as Bacillus and Mycobacterium species, will not grow. The converse is also true. By far, the vast majority of organisms (especially the enteric flora) belong to the group of facultative anaerobes. These are organisms which can grow in the presence or absence of most of the oxygen, Another small group, among which are a few medically important organisms, will grow only in the presence of a very low pO; and are called microaerophiles. An example is Campylobacter fetus, a gram-negative pathogen. The ability of an organism to grow in an atmosphere of reduced pO, may be used to selective advantage. As an example, the medically important facultatively anaerobic, gram + positive, hemolytic streptococci will grow at a reduced. pOs, while the growth of the generally contaminating aerobic forms will be repressed. In fact, these streptococci, when grown at reduced pO2, often produce larger colonies than when grown in air, an important observation, since the colony size of these organisms is generally minute (about 1 mm in diameter). Further, when grown on blood agar at reduced p02, the oxygen labile hemolysin secreted by many of these organisms often expresses itself more prominently, and the characteristic colonially peripheral zones of red cell clearing, or hemolysis, become more obvious. 16Convenient end commonly used methods for producing useful anaerobiosis include the “candle-jar", the use of commercial gas-packs, and the incorporation of non-toxic reducing compounds into the culture media, The “candle‘jar is prepared by lighting a candle in a jar containing cultures and immediately sealing the jar. The candle will burn until most of the oxygen is consumed and then go out. (The amount of trace oxygen left will inhibit certain obligate anaerobes). This "candle-jar" procedure is also recommended when an anaerobic atmosphere relatively enriched in CO is required for isolation of certain pathogenic bacteria. However, the inhibitory effect on bacterial isolation by candle exhaust has been demonstrated. Further, the oy ‘al percentage of CO; required for the isolation of some bacteria is above 5%, approaching 10%. This requirement is not always obtained in candle-jars, which may enrich the jar atmosphere only to 2-3% CO... Today, many leboratories use a commercially available packet to produce an oxygen deficient atmosphere, Immediately before use, water is added to the packet. The packet is then added to a jar containing cultures. The jer is sealed but may be equipped with a one- way valve. The chemical reaction produces either a gas which flushes out the oxygen or an alkaline product which absorbs oxygen fiom the chamber. Other methods for the growth of "anaerobes" do not require overt removal or displacement of oxygen. The approsch incorporates.a non-toxic reducing agent, such as thioglycolate into solid or liquid medium or 0.1.- 0.2% agar into liquid media. Ager, at this concentration, gives the liquid medium, a semi-solid consistency and reduces the diffusion of oxygen into the ligiid columin. Fluid thioglycolate broth medium, chopped meat medium, or iron milk medium are sometimes used for the cultivation of anaerobes. “Aerobic” organisms are readily grown on agar or broth surfaces in loosely capped vessels. Increased aeration of broth cultures is practically obtained by bubbling sterile gas mixtures through the liquid or by shaking. Tight closure of plastic screw ~ capped tubes can inhibit bacterial growth by preventing the entry of air. A special term, "pellicle", refers to the static membranous surface film of growth in "aerobic" broth culture. ‘The temperature of incubation may also be used as a selective control for the isolation of pathogens; however, it is not often practical. Organisms which "grow best” at teinperatures about 45° Care called "thermophiles"; others, and most of the human pathogens are in this group, . “grow best” at temperatures between 25 and 40°C, and are named "mesophiles" . Organisms which "grow best" at temperatures under 20 are called “psychrophiles", Generally, 37.5°C is the incubation used for the cultivation of human bacterial pathogens, 32 - 37°C for human viruses in cell cultures, and 18 ~ 25°C for the mold form of pathogenic fungi uvr =) Isolation of "Unknowns from Simulated Clinical Specimens" In some bacterial infections, the causative organism may be found in pure culture. This is often true in infections of the meninges or of the blood stream. In other infections, such as the throat or intestinal tract, the etfologic agent may be only one of several hundred types of organisms present, To identify an organism, it is necessary to obtain it in’pure culture so that its characteristics may be studied. Several methods have been devised for separating the organisms in a mixed population. One of the most frequently used methods is called "streaking", "strecking for isolation," or the streak plate method, In your application of this procedure, only one organism will be placed (inoculated) on a small area of a solid medium, made firm by a sulfated galactose: polysacchatide called "agar", which is a seaweed product that melts at 100°C but which does.not solidify until it cools.to 40 - 45°C. With an inoculating oop; the organisms. in:the: specimen. are.spread.over:the:surface of the agar-in a series of streaks. This eventually separates the individual cells or clumps:of bacteria from-each other. When the mechanically isolated cells multiply; they produce visible structures. called "colonies", Colonies are assumed to be pure .oultures of one organism. If the "founding" organisms. have been separated well enouigh, these colonies, presumably arising from a single cell, do not touch each other, If colonies are too close to each other, they will become confluent as they grow, eventually producing a mixed colony. In this case, it will be necessary to restreak the mixed colony on another agar plate to separate the individual organisms. ‘The purpose of this exercise is for you to identify the genus and species of the bacterium in your unknown, This is essentially a *practice” unknown, 18Te Exercise: ‘A. Streaking of plates (watch instructor's demonstration), 1, Today each student will make at least two different streak plates using their "unknown" specimen, The specimen contains only one microorganism, 2. Team up in three groups of 5 - 6 members: Team A, B, or C, Remember your team letter, Each team will receive 5 - 6 different unknowns, They are each labeled with team letter, and whether they are cerebrospinal fluid (CSF), stool (S), throat (I), or wound (W), Everyone's unknown is different. We used 10 different. bacteria in this ‘exercise, You will be asked to share all your results with everyone in your team. This is 2 Steam" project. You will need all your team's information in order to successfully ‘identify your unknown, . "Watch your instructér’s demonstration of streak plating before doing any of your own ‘work. There areextra Petri dishes filled with sterile Trypticase Soy Agar for practice. ‘Practice’on these plates before you begin to examine your unknowns, Use tap water, or ‘water left on your lab bench as a specimen, 4, Select and sign for any unknown specimen-assigned to your team. Place it in a test tube rack at your work bench, Carefully note the nature of the specimen: CSF, S, T, or W. 5. Select the appropriate media, You must use the right media or this Exercise may fail. 6, If you choose a "CSF* specimen, take only one blood (Hb) and one chocolate agar plate. [f you choose *S" (stool) specimen, take ofly one MacConkey Mac), one Hb and one trypticase soy agar (TSA) plate. If you choose a "T™ (throst) specimen take two Hb plates one for aerobic growth and the second in a candle jar. Tf you choose a "W" specimen, take only one each of Hb and TSA late, Do not open these plates unti! you are ready to "streak" them, following the instructor's demonstration. 7. The process of plating and streaking is performed with one edge of the Petri dish lid just raised to permit entrance of only the specimen and inoculating loop. The dish lid should not be entirely raised or put down on your bench top during this procedure. 8. Streaking the specimen (Figure-1). Read the following instructions before watching the demonstration and streaking your own plates. Students who chose the throat specimen should streak their two Hb plates with the same material, 19=» Tr fe ISOLATED COLONIES \tme 4 . Transfer a loopful of specimen, with a sterile cooled inoculating loop, as demonstrated by the instructor to a spot on the medium marked "I" in figure 1. Spread the loopful of material evenly over Area I as demonstrated. . With a sterile inoculating loop "stab" (undercut) a portion of the inoculated area of the plate so that the organisms present are buried within the:blood agar. Tum the plate about 90°, Without flaming the loop, streak a small portion of the invisible surface deposit on Area I across an unused portion of the medium (Area II). The streaks should be very close together; work toward the center of the plate. Cover the entire Area Tf; do not leave any unsmeared surface. In this step, another 1/4 to 1/3 of the plate should be covered. The loop is then flamed and allowed to cool for a moment. 20eas) 4d. The plate is tumed about 90°, and the third set of streaks is made, Begin at the edge of the plate; smear a small portion of the invisible surface deposit taken from Area Il (as above) across this new Area II. The streaks should be very close together; work toward the center of the plate. After this, the entire plate should have been "streaked" (smeared). Flame the lodp and put it down; you are finished. The whole process should take about ‘two minutes. Figure 1 shows an additional streaking step in 4, this can be optional. Some bacteriologists flame the loop between Areas I and II or between Areas II and III. ‘They then “pick - up" some deposit from an carlier area and transfer it directly to the next, ¢. Label the medium - containing dish with your name, date end name of organism or specimen. Top lids may be accidentally switched and should not be the only part of the Petri-dish labeled. f. Place,the Petri dishes containing Mac and TSA in the 37°C laboratory incubators in an invertéd position: Inversion prevents water of condeisation froin falling onto the surface of the agar. If the surface becomes moist or wet, some bacteria will grow in a confluent sheet instead of in distinct colonies. Inversion is unnecessary at room temperature. If you chose a throat specimen, you were to do duplicate streak plates on Hb ager. Incubate only one of these Hb plates in the 37°C incubator as the others. The duplicate plate is to be handed in to the instructor. The instructor will incubate this Hb streak plate in a "candle ~ ja": Normally, all blood and chocolate agar plates are incubated in a reduced pO2 environment, ike acandle-jer. g. There are some useful variations to this procedure, The above recommended sequence ‘seems to be best when speed is not a concern,a) Note}. Make sure that the loop at the end of your inoculating instrument is flat, is closed, and set about 10° from the shank Note 2. Itis not always necessary to stab streak plates. We do it here because we are looking for certain facultative anaerobic bactetia, whose distinctive cultural properties are better displayed when grown in a lowered pO2 beneath the agar surface. Specifically, the production of the somewhat Op labile B - hemolysin of Streptococcus pyogenes Group A. Additional Notes: 2 |Exercise VI The Gram Stain /the Acid Fast Stain ‘The Gram Stain In the clinical setting, bacteria are usually observed by light microscopy. There are several different techniques of light microscopy, including transmitted, phase contrast, fluorescent and darkfield microscopy. Stains are usually used to facilitate the observation of micro-organisms. ‘These dyes are used as their salts that dissociate into positively and negatively charged ions. Dyes are classified as acidic or basic, depending upon whether their chromophore is a negative or positive ion, respectively. Bacteria carry a net negative charge at pH 7, end the chromophore of most dyes used for microbiological staining is a positive ion. Such dyes are called basic dyes. The most comménly used are crystal violet, sofranin, methylene blue end basic fuchsin. If only a single stain is applied to'a bacterial smear (e.g. aqueous erystal violet, 1 %), the method is referred to as simple staining. This procedure is used.to observe the morphology and arrangement of the organisms, However, there are more complicated staining procedures which are of greater aid in the differentiation of bacteria. ‘The most frequently used is. the Gram Stain, perfected in 1884 by the Danish physician Christian Gram, The Gram Stain is a differential stain, Depending on the technique, there are three or four steps in the Gram Stain procedure. First the smear is stained with crystal violet, then by the application of Gram’s iodine (Lodine in KI). Next a decolorizer (alcohol or acetone- alcohol) is applied, and in the ‘final counterstained step, safranin is applied. Some bacteria will retain the crystal violet - iodine complex, appearing blue - violet - black in color, and are called gram - positive, Others will be decolorized and. will be counterstained red with saftanin, these appear pink or red, and are called gram - negative. From recent evidence, it appears that the alcohol or acetone - alcohol mixture alters the wall of gram ~ positive organisms, The altered wall traps the insolable crystal violet - iodine complex. ‘These changes do not occur in gram — negative organisms. Gram - positive organisms differ from the gram - negatives in other respects. These include susceptibility to drugs, including penicillin (gram - positive organisms are generally more susceptible to penicillin), the composition of their cell walls, and susceptibility to killing by immune antibodies, and to inhibition by various dyes. Some caution has to be observed when interpreting Gram Stain reactions because gram - positive organisms are frequently over - decolorized and appear falsely gram - negative. False gram - negative reactions may also be related to age, growth medium, and pH. As gram - positive cells age and / or the pH of their growth medium become acid they tend to stain gram - negative, 23‘The 3- Step Gram Stain of Difco. The 3-Step Gram Stain is a new (1995) procedure, It was developed to permit more control over the decolorization step of the traditional 4 step technique (Appendix I). Over - decolorization is difficult to prevent. The solvents used to decolorize, acetone and alcohol, work relatively fast (1- 3 seconds), and in 2 wamm laboratory, the time to over ~ decolorize is almost equal to the minimum time it takes to perform the step. ‘The 3- Step Gram Stain uses an ethanol based decolorizer of unknown proprietary composition, Importantly, it lacks acetone, commonly used in the usual alcohol ~ acetone (I :1) decolorizer. Therefore, besides offering better control of decolorization, it is less toxic and considerably less flammable. There are many other modifications of the Gram Stain procedure, One of the most valuable is the 4- Step procedure, and is the’ technique you will probably find still used in most hospital laboratories. It is described:in Appendix. I. ‘Another: particularly: valuable and. simple. modification‘of the Gram Stain procedure is the addition of a few drop of 5% (w/v) aqueous sodium ‘bicarbonate (household baking soda) to the ‘crystal violet step. The bicarbonate buffers: the-stain; solution and:reduces'the number of false gram’ - negative reactions that occur when the smears are acid. Acid smears are-assoviated with pus. Gram ~ stains of wound specimens should be buffered. The debris from lysed WBC is generally acid, The-3- Step ™ Bacto - Gram Stain procedure that you will use is as follows: 1. Use air-dried, gently heat fixed smears, 2. Flood slide with Bacto Gram Crystal Violet for 1 minute. Then, reverse slide, place under gently flowing cold tap water to wash off excess dye. 3. Flood slide with Bacio Gram Iodine for 1 minute. 4, Wash off iodine using Bacto 3-Step, Gram Safranin - $, Flood and. stain for 10-20 seconds. Wash off excess dye as.in Step 2. Gently blot dry in Bibulous Paper Pad or air dry. Examine microscopically using: immersion oil and the oil immersion: objective of the microscope. Gram - stained bacteria are dead. Discard stained smears in the paper trash. Unstained bacteria are alive and dangerous. Discard unstained smears in the autoclave-pans. The assessment of the Gram Stain reaction in any given preparation is based on the staining reaction of the majority of the cells. The reaction at the periphery of the Gram stained smear is considered by some bacteriologists to be most reliable, 24Grams stain ex 1 ise: Preparation of smears for staining a Share cultures. Obtain one prepared 18hr TSA slant of Escherichia coli and Staphylococcus aureus Using a loop, place a drop of water on a clean microscope slide. Use a sterile inoculating needle to transfer a very small.amount of bacterial growth from the ‘TSA slant to the drop of water, as demonstrated by your instructor. Being very careful not to contaminate the slants, first use S. aureus for one slide, then E.coli for a second slide, Mix each of the organisms in the water until a very faint, just visible cloudiness detected. Bum the excess growth off the loop or needle (avoid splattering), On the third slide, put a smaller amount of both bacteria in the same drop of water. Allow the smears to completely air dry. ‘With cere you can put all three smears on the same slide. Mark the underside of ‘your slide with three circles. Make your smears on the slide within the circles. = Make more: than one slide’ in case your fi'st attempt at Gram ‘staining vis unsatisfactory. You will save time by having smears ready for staining. Do not heat the slide(s) in order to dry the suspensions, Let them air dry, With the dry smear side up, pass the slide through the bumer flame two to three times to fix the smear to the slide, The slide has to be warmed and not scourged to allow for good fixation: step. Heating will fix the cells by denaturing the proteins and adheres them to the slide.. Remember, until stained the organisms in the dried heatfixed smears are viable and, therefore, dangerous. Using air-dried and fixed preparations, Gram stain the microorganisms according to the Gram stain procedure. 25a The Acid - Fast Stain Some species of bacteria. (Genus Mycobacterium) do not stain by simple staining procedures. Staining of these organisms is facilitated by application of heat, even when treated with a decolorizing agent such as acid alcohol, they retain the dye. They are designated as acid - fast organisms. This procedure employes initial treatment with carbolfuchsin followed by acid aclohol and methylene blue. Acid - fast organisms are not decolorized and appear red, the none acid - blue. fast organisms are decolorized and counter stain by the methylene blue, hence they appear Materials: > Organisms: Nutrient - agar slant cultures of Mycobacterium smegmatis and Staphylococcus aureus. ~ Staining solutions: Zich!'s carbol fuchsin, acid alcohol and methylene blue. ~ Strip of blotting paper. , - Egg albumin or serum, Prepared acid - fast stained smear of tuberculosis sputum. Exercise: moe Smeer should be prepared in a small drop of egg albumin or serum, The protein enhances the adherence of the bacteria to the slide and also provides material for light background staining. BYERS a2 Prepare and fix smears of i, smegamatis and S. aureus. Cover the'smears with a strip of blotting paper. Saturate the paper with Zieht’s carbol fuchsin, Heat the slides to steaming, Do not allow the slides to dry. Allow the staining to continue for3 - 5 minutes, and remove blotting paper. Rinse with tap water and decolorize the smears for 10 to 30 seconds with acid alcohol. ‘Wash the slides with tap water, Apply methylene blue for 30 to 45 seconds wash, blot dry. Examine the smears under oil- immersion objective. Acid — fast organisms stain red, and non acid - fast organisms stain blue. Record the color of organisms in each preparation, and indicate their acid - fast reaction, 26‘The Acid Fast Stain Demonstration ‘The causative agent of humen tuberculosis, Mycobacterium tuberculosis, is difficult to stain by most procedures: including the Gram Stain. Robert Koch, who described the organism in 1882, developed a successful staining technique to visualize M. tuberculosis. His original procedure has been frequently modified and today the modification often used is the Ziehl-Neelsen (Z-N) method. The Z-N method uses first a solution of hot carbol fuchsin (a mixture of phenol and the red dye fuchsin) to stain the organisms. The stained smear is subsequently decolorized with a solution of dilute sulfuric or hydrochloric acid in 95% ethanol (“acid-aleohol”). Methylene Blue is often used as a counterstain. Acid-fast M. tuberculosis and other Mycobacterium spp. will stain red-crimson-magenta, most other bacteria and cells will stain blue. The acid-fastness of M: tuberculosis is associated with the integrity of the cell wall and its content of rather firmly bound lipids. ‘The phenol apparently aids in the penetration of the fuchsin dye. After the carbolfuchsin step, most bacteria will be thoroughly stained, The unique property of the acid-fast genus Mycobacterium is their resistance to decolorization by the acid-aleohol. Hence, Mycobacterium retain the dye while almost all other microorganisms are decolorized and will subsequently "take" the methylene blue counterstain. Microscope demonstration. Examine the acid-fast (AF) stain of Mycobacterium tuberculosis in sputum from a patient with pulmonary tuberculosis 2Labs 5 & 6 Work Sheet Part 1: Isolation of bacteria (streak plating) Part 2: the Gam stain / the acid fast stain Part 1: Objectives : 1, To leam about different types of meidia; simple, complex, selective, and differential. 2, To study the growth characteristics of 10 microorganisms on different agar media 3. -To master the technique of streaking and aseptic transfer 4. To lear how to isolate a pathogen from a mixture of bacteria 5. To become familiar with:common:pathogens to humans. 6. ‘To become familiar with different-types of hemolysis on blood agar. ‘Materials: 1. 10 organisms each grown on 4 different agar media: Blood agar, (BAP) chocolate agar (choc A), trypticase soy agar (TSA), MacConkey agar (Mac) 2. Unkown broth culture as clinically simulated specimen CSF, stool, wound, throat. 3. Bacteriological media: CSF /B.A, choc A Stool B.A, TSA, Mac Wound /B.A, TSA Throat /B.A, B.A 28Results: 1. fill in the table Growth characteristics of bacteria 2. your unknown number or letter 3.-your unknown results Specimen Blood | Agar | Chocolate Agar MacConkey Ager Trypticase Soy Agar . Escherichia coli Moraxelli gatarrhalis Pseudomonas aeruginosa . Salmonella species . Shigella species . Staphylococcus aureus Staphylococcus epidermidis . Streptococcus pneumoniae9. Streptococcus pyogenes 10. Streptococcus viridans 11. Candida albicans Unknown, Report inthis table: Growth; slight growth or no growth. Examine for hemolysis on blood agar, pigmentation, opacity, surface.texture,-mucoid-or not, smooth or rough surface, any other features, 30Discussion I What was your unknown, give the reasoning for your conclusion. What is the difference between selective & differential media? Give examples of each. . Which organisms produced pink colonies on MacConkey agar, Explain? You have incubated some of the plates in a candle jar, explain why ? Which is easier? To isolate a pathogen from stool specimen or from CSF specimen & why? 31. Discuss different pattems of hemolysis on blood agar? What is producing this hemolysis? . From the bacteria you used which one causes: Pharyngitis: Urinary tract infection: ‘Wound and bum infection: Bloody diarrhea: Abscess and skin infections: Endocarditis in intravenous drug users: Endocarditis after dental extraction: Oral or-vaginal thrush: 327 Exercise VII Growth Characteristics of Bacteria ‘When mechanicelly isolated bacteria are grown on the surface of ager media, they form aggregates or colonies after repeated cellular division. Colonies may eventually be composed of billions of cells becoming visible to the naked eye, and range in diameter from few tenths of one millimeter to several millimeters, or ocoasionally even centimeters. The development of visible colonies is often very rapid, Some. common bacteria will produce visible colonies within a few hours of growth on blood agar at 37°C. In any event, if the colony arose fiom one cell, it is safe to assume that it is composed of copies of only the same microorganism - a pure colony. Some colonies may be composed of more than one type of micro - organism. Such mixed or impure colonies can arise in at least two ways: if, after streaking, two different types of bacteria are "stuck" together, or if different adj colonies become conflueit duting their growth. It is gerenally wise to check the purity of colonies. by doing a Gram Stain of a small portion, If the colony is pure, only one type of bacterium. will be seen on the Gram stained smear. When adjacent colonies are almost touching, it is best to assume that they have "mixed". Do not consider them "pure" even though their extreme edges appear to be unique and not intermingled. ‘You may be tempted to use such colonies without the delay of preparing another streak plate; it is generally an, unwise judgment. Even when apparently identical colonies are confluent it is best to assume that the mixture is not homogeneous. You should only attempt 10 study isolated colonies whose purity has been confirmed by the Gram Stain, Critical examination of colonial morphology is an overlooked exercise. There are many facts about colonial morphology which are. of great help in distinguishing, identifying, and determining the number of different organisms growing on agar media. These often diagnostic signs are only leamed by the experience of close, very close, scrutiny, and practice, and constitute one of the real arts of microbiology. To reemphasize, in addition to being wrong, it would be naive to believe that a bacterium has an identical colonial appearance, or even a smilar one (if it grows), on different media. Obviously, one would then believe that there is more than one organism under scrutiny. In fact, the organisms you will study, almost without exception, demonstrate discernible differences in colonial motphology on all supportive media which will become available to you. Even on the same streak plate, the colonial morphology of an organism may vary. Close scrutiny of your streak plates may also reveal less obvious features or tiny colonies, "miniaturized" by poor steaking. You will observe that colonies in a crowd" are more antagonistic to each other, i.e.. are smaller than those which are more distantly separated. This size limitation is thought to be due to the accumulation of metabolic inhibitors rather than a fall in the pH or a depletion of nutrients. 33Today's exercise is a demonstration that will provide you with the necessary data to identify your unknowns. The demonstration consists of Hb, Choc, TSA and Mac media showing the appropriate growth responses of the same ten organisms that were given to your team in unknown mixtures in exercise I. Bach plate is inoculated with only one of the organisms listed in table 1, The demonstration includes an uninoculated plate for comparison. Determine which medium or media supports the growth of each organism. Write your observations in the table at the end of the exercise. Report growth responses as: no growth, slight growth, or growth. ‘The components of cach of the four’ media are listed in Appendex III. Can you relate any medium characteristics or components to the suitability of the medium to support or distinguish bacterial growth? Do “simifar" bacteria respond similarly? Especially study the reactions occurring on MacConkey’s Agar. Which medium is selective? For which bacterium? Which medium is differentiating? For which bacterium? ‘There are many prominent and subile differences:in'bacterial growth patterns thet are very useful in distinguishing microorganisms from-each otherFor our needs, in addition to the ability of the medium to:support grovyth or not;.the ‘presence: of hemolysis.on blood agar, the presence of any pigment, and the opacity-of the microbial growth:are useful. discriminators. Your instructor will discuss all of these-features with you. Hemolysis-is‘of most importance. ‘There-are three types'of hemolysis-that are distinguished in the clinical microbiology lab: alpha or beta hemolysis. No hemolysis is sometimes called "y hemolysis", Alpha hemolysis is a partial clearing of the blood surrounding the colony and is en indication of the production of methemoglobin. The a-zone looks greenish or brownish. Under magnification you will see remnants of RBCs in a-zones. B- hemolysis.is.a complete clearing of the blood surrounding the colony. In this case the RBC membrane is usually completely dissolved. You must be able to, identify and distinguish B- hemolysis: ee Examination of Streak Plates Exercise: 1, Select a pure colony (containing: only copies of one organism) and restreak on a plate of the appropriate medium. How do you know the colony'is pure? 2, The choice:of medium is important: Generally, you would restreak’the organism on the medium. it grew best. However, if it:grew:well on MacConkey’s restreak on TSA, Blood or Chocolate medium. Why is this advisable practice in a clinical situation? 3. Label plates carefillly. Incubate inverted at 37°C. All blood and chocholate agar plates should be incubated in a "candle jar." 34Exercise: If your "unkown" streak plates show well - isolated colonies, prepare Gram stains from these colonies. Correlate the gross appearance of the colonies and the Gram reaction and morphology of their cells, as viewed after staining, ce Additional Notes: pGrowth Characteristics of Bacteria on Different Media Blood ‘Chocolate racConkey’s [Trypticase Soy Agar Z. Escherichia coli 2. Moraxella catarrhalis 3. Pseudomonas aeruginosa 4 Salmonella species 5. Shigella sonnei Staphylococcus aureus 7. Staphylococcuspidermidis. 8. Streptococcus pneumoniae. 9. Streptococcus pyogenes 10. Streptococcus viridans 11. Candida albicans Report in this Table: Growth, Slight Growth or No Growth. Examine for hemolysis on Hb, pigmentation, opacity, surface texture, mucoid or not, smooth or rough surface, any other features. 36Exercise VIL Antibiotic Sensitivity Tests To effectively manage a patient with infection, a physician must Jnow and have knowledge about: 1) the causative agent; 2) the drags, in this case, the antib&ZS that might be used; and 3) the patient's Aistory, physical exam, and laboratory findings, The clinical microbiology laboratory is involved in these considerations since it provides, specifically in our case, information as to the probable etiological agent, the antibiotics the organism is susceptible to, and the degree of susceptibility of the patient's isolete(s), as well as, if treatment has started, the antibiotic levels that have been achieved in the patient To manage. the patient, the physician should also know: 1) the general characteristics of me infectious agent and the special or peculiar pathological processes that follow infections by this microorgantsin; 2) the pharmacological properties of the antibiotic(s), including toxicity, protein binding, distribution, absorption, and excretion; and 3) the immune status of the patient and any other modifying factors, e:g., the presence of renal dysfunction that may bé exacerbated by any proposed therapy with known renal toxicities. After isolation and identification of the probable etiologic agent from the patient's specimen, the Jab will often routinely, without a specific request, test the Isolate for its antibiotic susceptibilities. As a rule, identification of an organism and its sensitivity may be reported as early as 2. nus after the specimen is received. In some cases with slow growing bacteria, complete identification to genus and species and antibiotic sensitivities may take several days. - Organisms’ often have characteristic susceptibility patterns to a battery of different test antibiotics. These patterns can be used with caution as an aid to identification just as the Gram stain is used Sensitivity results may suggest the most rational choice of therapeutic agent even without knowing the organisms identity. For example, only knowing that you are dealing with an unidentified gram-positive cocous that is resistant to penicillin G, may be an extremely valuable, even life-saving, finding, Many microbes quickly become resistant to antibiotics and the frequency with which they are encountered may be related to the frequency that the antibiotics are prescribed in the community. Jn this circumstance, bacteria may be encountered that were previously understood to be sensitive to a certain chemotherapeutic agent but have now become resistant. Antibiotic resistance may occur in patients very quickly after therapy hes begun. Occasionally, after a day or two of therapy (but sometimes within hours) resistant organisms may be detected in your patients and become a part of their dominant flora. This rapidly acquired resistance and the presence of resistant forms in hitherto susceptible microorganisms in your practice requires, continued surveillance. 7q Ee ‘This section and Exercise on Antibiotic Susceptibility Testing is designed to indicate what tests are available to you in the clinical lab, how they are performed, and to describe their interpretation, It includes a brief discussion of the newest innovation in antibiotic sensitivity testing, the E-test .. In practice, the three most commonly encountered types of antibiotic sensitivity tests are the 1) Minimum Inhibitory Concentration (MIC), 2) the Minimum Bactericidal Concentration (MBC), or Minimum Lethal Concentration (MLC), and 3) the Kirby-Bauer or Disc Diffusion test. A micro-modification of the MIC test is available (MIC by micro test). There are two other related tests using the patient's serum: 1) serum assay test, and 2) the Antibacterial Activity test (ABA). Finally, there is the somewhet revolutionary E-test that will be demonstrated in the laboratory . 1) Minimum Inhibitory Concentration (MIC) ‘The oldest'standardized:method developed for determining antibiotic. sensitivity is the Minimum Inhibitory Concentration Test (MIC), also called:the tube dilution miethod. In this procedure, the antibiotic: is “diluted: serially in broth in ‘a-set.of ttibes. ‘The tubes are then inoculated with a standardized ‘amount: of the isolated -organisin. After incubation overnight, the tubes are examined. for turbidity with the naked-eye;-clouidiness in the ‘broth indicates growth,-whereas clear tubes indicate no growth. The MIC is defined as the minimum concentration of antibiotic that will inhibit the growth of the organism {Fig. 1). This test can be miniaturized. 25 62 31 16 08 04 02 0 Hg antibiotic /ml Fig. MIC (MIC = 3.1 pg/ml) 2) Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC) Although the MIC is generally considered to provide the most useful information on sensitivity, occasionally it is desirable to know the amount of antibiotic necessary to actually kill the organism rather than just inhibit its growth. In this case, broth from the MIC tubes is incorporated into cooled; liquefied agar ‘2.2 is poured into plates and allowed to solidify. After 18-24 hours of incubation, the plates are examined, and the lowest concentration of antibiotic that shows no growth is called the minimum bactericidal concem-tration (MBC) or minimum lethal concentration (MLC) (Fig 2.) 38al Noenfibiotic Noinoculum Neal 42 f# = ANG tk EAR RSS AST | Fig. 2, MBC (MBC 1/4) Both. the MIC and MBC tests aid in determining the levels of antibiotics which-must-be achieved in the body fluids to affect the probable etiological agent. Interpretation must be tempered by the realization that this model, as well as most others, is incomplete because of the presence of additional factors that are Involved in vivo (such as protein binding and the defense mechanisms of the host) that cannot be tested in this procedure. We must also be aware of the concentration of the particular antibiotic that is achievable in the blood, tissues, or urine depending on the route of administration. In general, the clinical dosage at 2-3 times the MIC is often used in treatment, Pharmacological considerations are very relevant, To illustrate, the hypothetical organism used in the example above had an MIC of 3.1 g/ml. If an effective clinical seriiia-level is 6 to 9 g/ml (2-3 times the MIC), but the intended oral medication results in a serum goncentration of 1 to 3 pg/ml, there is some expectation that treatment would be inadequate. If, however, intravenous therapy were to be used with a dosage that results in non-toxic serum concentrations of 40 to 50 pg/ml, there is less doubt of treatment success.f The value of MBC's has been questioned, since MBC’s are more variable with minor changes in test condition (and are therefore less reproducible), MBC's vary more for different strains of the | same species, and are of less value in predicting clinical responsiveness than MIC's, Despite these problems, antibiotics have traditionally been divided into two categories, "Bacteriostatic" antibiotics generally have MBC's much higher then MIC's for the majority of sensitive organisms, and the MBC's may not be surpassable by serum concentrations achievable with clinically useful dosage. "Bactericidal" antibiotics generally have MBC’s which are the same as or only slightly, higher than MIC's for the majority of sensitive organisms and may be easily surpassed by serum concentrations achievable with clinically useful dosage. Example: Achievable -Achievable -. Achievable MIC MBC “serum conc. @mi) (emi) (g/ml) Antibiotiex. ; 31° 50 25 (bacteriostatic) Antibiotiey 3 62 25 (bactericidal) Most likely, both antibiotics will be effective, however, many individuals fee! thet the use of "bactericidal" antibiotics is preferable to the use of "bacteriostatic® antibiotics, especially for such infections as bacterial endocarditis, for transplant and cancer chemotherapy patients. 3) Kirby-Bauer Dise Diffusion) Perhaps the:most common test for the determination of antibiotic susceptibility is the disc diffusion test. The Kirby-Baver. method is'a highly. standardized form of this-test which takes into account properties) of the-antibiotics, the organisms, and the agar medium in which the test.is performed: Briefly, in this test, ‘small. paper:discs are: impregnated with known amounts of antibiotics.and aseptically placed on:agar plates previously seeded with a known concentration of the organism: ‘The drugs diffuse from the discs into the agar at rates which are characteristic for each agent. This resulis in a concentration gradient around each dise, with areas closest to the disc having the highest concentration. As the lav of organisms grows, zones of inhibition appear around the discs, The zone size (linear thickness) around a particular disc is directly related to the log of the MIC of that antibiotic for the organism being tested. Essentially, the smaller the zone size, the higher the. MIC, and, therefore, the less susceptible an organism is to that particular antibiotic. 40a ‘However, many factors affect the zone size and must be taken into account. So-called “critical zone" sizes have been established for cach antibiotic, based on a relationship between zone size and MIC, the antibiotic's diffusibility and solubility, its toxicity to the host, protein binding in Viveeine, absorption by tissues, rate and route of excretion by the host, etc, An organism exhibiting a zone of inhibition larger than adopted critical size is considered susceptible, while one showing smaller zone size is classified as resistant, Naturally, zone. sizes indigating resistance and sensitivity are different for each agent. For example, gentamicin levels above 12 g/ml in the blood are toxic to the patient, so organisms inhibited at this range or above are considered resistant. The effective dose is an inhibitory level we cannot safely achieve in the patient, Organisms sensitive to 3-4 ug/ml or less are considered to be sensitive (remember that 2-3 times the MIC is often used as the therapeutic range). Microbes falling in between these points are intermediate in their susceptibility. Using the regression line for gentamicin (Fig.3), zone sizes are found that correspond to these ranges. ‘ganisms can be reported out as resistant, intermediate, or sensitive based on the size of the zoné of inhibition SYAYE the antibiotic disc. 08: 6 ou (2) 30 : ik Diameter of zane of Inhibition (mm) Fig. 3. Gentamicin Susceptibility (Resistant, Intermediate, and Susceptible) Note: Each dot represents the response of a different microbe to various MICs of gentamicin. In this experiment we may conclude that dise zone of inhibition of more than about 15 mm means tha the microbe Is sensitive o gentamicin ara ic Certain technical factors also affect zone size. These include the concentration of antibiotic in the disc, the agar concentration and the depth and the pH of the medium, and the atmosphere of incubation. The Kirby-Bauer method represents a successful attempt to minimize variability in test results occurring from day to day and from laboratory to laboratory due to these factors, Briefly, organisms are transferred oa tube of trypticase phosphate, tryptic soy, or Mueller- Hinton (MH) broth, Afer brief incibation (2-S hours), the resultant broth Eulture is diluted to a specific turbidity-(equal to about 19" cells/ml}, A small aliquot of this diluted cell suspension is uniformly spread over 80 ml of Mueller-Hinton agar solidified in a 150 x Smrn Petri dish. After inoculation, these plated are briefly dried, and no more than 12 antibiotic disc of prescribed potency are placed on the surface in a standard pattern. These plates are incubated for about 16 hours at 37°C, After incubation, the zones of inhibition are measured with a ruler or with a special Single Dise Zone Chart-(Kirby-Baver)...Each .chart has specific. circle patterns, associated with specific antibiotics: There issat least one:pattern'avaiilable foralmost ‘all;clinical-antibiotics. The Petri dish: is placed oversthe:chait:so that the disc. is.centered:on‘the:center of its designated pattern. For instance, to:meesure the vorie'srourid.a"PenicillinG-dise.(P), you must use. the P pattern, or chloramphenicol C, streptomycin’S-etc; ‘Each fattem ‘has "an, identified center which is the exact sizeof the:antibiotic disc edge andthe first ring is the resistant. zone.(R), the zone. between the two outer rings is the intermediate sensitivity zone (I), and outside the outer ting is the sensitive zone (S): If the outer edge of the experimentally determined zone of no growth falls in the S area of the appropriate pattern, the organism is sensitive to thet antibiotic (Fig.4). Examples: OO a@ Penicillin Penicillin chloramphenicol R = zone of resistance I S= sensitive intermediate zone of resistance Fig. 4. Different Grid Patterns for Determination of R, I, 8 42Some antibiotics have two disc patterns to choose from, e.g., penicillin G has PI arid P2. The choice between these two is dependent on whether the organism being tested is a staphylococcus; if it is, use Pl, and if not, use P2, Examine these two choices on the accompanying figure. What does this indicate about the relative sensitivity of staphylococci (o penicillin G? Remember that results are reliable only if utmost care is exercised in the standardization of the method (including testing the potency of the disc being used), Otherwise, there are too many variables for results to be meaningful. 7 4) MIC and Identification by Microtest There are microtest plate methods for bacterjal. identification and determination of antibiotic, sensitivities, Bacterial identification is accomplished by conventional biochemical tests aid antibiotic sensitivities are determined by the.Misimum Inhibitory Concentration procedure discussed ptéyiously. The main difference between the MIC by microtest system and the conventional-test tube for bacterial identification and antibiotic sensitivities is that the microtests are performed in the small conical wells of plastic microtest plates rather than in test tubes. Miniaturization outs expetses in ternis of media cost; however, it also permits“an increase in the number of biochemical tests which can be performed on each potentially pathogenic organism isolated from the patient, Unusual organisms that require three or more days for definitive identification by the conventional test tube method can often be identified in 48 hours using the Mirotest method. 5) Serum Assay ‘The serum assay tests for the amount of antibiotic actually in the patient's body. The procedure is usually performed on serum although other body fluids, such as spinal fluid or urine, can be used. Assays are generally requested for two reasons: 1} fo determine whether or not a patient is receiving doses of antibiotics adequate to treat their infection; and 2) to monitor the levels of toxic drugs. In this procedure, an agar plate is seeded with a standard amount of an organism of known sensitivity to the drug to be tested. Paper discs are then impregnated with known amounts of the antibiotic (in a decreasing range) and are placed on the plate along with a-disc containing a known amount of the patient's serum. After incubation, the zones around each’antibiotic disc are measured and a curve is drawn relating zones of inhibition to known amouiits of antibiotic. The zone size around the patients serum impregneted disc is also measured and compared to the curve to find the corresponding antibiotic concentration, 436) Antibacterial Activity (ABA) ‘The antibacterial activity assay, or ABA, is essentially a’ combination of the MIC test and the serum assay test. In this procedure, the patient's serum (or other body fluid) is serially diluted in a series of tubes, and each tube is inoculated with a standard amount of the infecting organism isolated from the patient. The tubes are incubated for 14-24 hours and examined for turbidity. UUUSGGG8 0 1/4 18 IMG 132 1164 1/128 Dilutions of patient's serum Fig. 5. ‘Detennining the ABA (ABA= 1:4) Dilution of the patient's serum will result in-a.corresponding dilution-of the-antibiotic that is:present, ‘The “highest ‘dilution “of serum-whichowill visually inhibit growth of the organism: is considered the ABA:An‘ABA"of 14’isidesirable since this indicates-that the serum contains about four times the amount of antibiotic needed to inhibit the organism (or four times the MIC) (Fig. 5). 7) The E-Test As the previous discussion indicates, there are several methods for antimicrobial susceptibility testing. In addition, there are automated or mechanized techniques besed primarily on some of these standard methods. Many of these tests are used only qualitatively. As resistant strains of microbes become more prevalent, the choice of therapeutic agents becomes limited, Concomitantly, quantitative information gained by knowledge of MIC’s and MBC's, or from other assays’ that we-have not discussed in this: Exercise, such as killing-curves and post-antibiotic effects; become. clinically’ valuable in. rational treatment.” Particularly. in instances of.life threatening infections, such as - bacteremias:- and. meningitides. where pharmacokinetic-and pharacodynamic. data are-valuable. “Although, frequently requested and performed in the clinical laboratory MICs and MBCs, as examples of quantitative techniques, are considered by some to be cumbersome, time consuming and not highly reproducible. There is some need for a simpler and accurate antibiotic susceptibility test. One response to this need has been the development of what is now called E- test technology. 44‘The E-test (AB BIODISK, Solna, Sweden) uses a non-reactive, impermeable plastic strip (50 x 5 mm) that is impregnated on one side with a predefined antibiotic gradient, The gradient generally covers a continuous MIC range corresponding to 15 two-fold dilutions. The gradient values are visibly numbered on the reverse side of the strip. Over 100 antimicrobials are now available in the product range for testing of aerobic bacteria and fastidious orgenisms* such as Pneumococci, Haemophilus, H. pylori, Meningococci, Gonococci, Anaerobes, Fungi and Mycobacteria. (Fig. 6) Ini practice, the test organism after dilution to a standard turbidity is spread on a Mueller-Hinton ager plate to produce confluent growth. The. E-strip is aseptically applied to the dried inoculated agar surface and incubated overnight. ‘The antibiotic rapidly diffuses from the strip to produce a continuous gradient of antibiotic injthe agar. After incubation, the MIC is read from the test strip at the point where the elliptical zone of inhibition intersects the strip (Fig. 7). “The’ E-test’s role in the clinical assessment of a drug's valuc.is almost unlimited," says'M. L. Sanchez anid R. N. Jones in, The:Antimicrobic Newsletter 8:1,1- 8, 1993. At present, the major drawback of the E-test is the high cost. 45 Fig. 6. E-test Strip. Diagram of an B-test strip (5.x 50 mm) with antibiotic gradient, Fig. 7. E-tests on a strain grown in a confluent sheet. Note various growth responses to different antibi Note the appearance of resistant colonies in the E~ test at -o'clockLab 8 Work Sheet Antibiotic sensitivity Testing Objectives: 1, To introduce the three most commonly used antibiotic sensitivity tests, MIC, MBC, and-the disc diffusion test. 2. To:become’familiar-with snew’:terms.as'the:minimum inhibitory concentration, ‘minimum: bactericidal-:concentration, .bactetiostatic ‘and. bactericidal, resistant, intermediate, susceptible. = 3: Torperform the disc diffusion test (Kizby- Bauer test). 4, To become familiar with: - Serum assay test - Antibacterial activity test ~ E-test Materials: 1, 2Broth cultures of B. coli & S. aureus 2. 2 Mueller Hinton agar plates 3. Antibiotic dises 4, Demonstration of MIC test;.MBC test 46Results: Fill in the table showing the diameter of the zone of inhibition in mm. and the result of the sensitivity of the bacteria compared with the reference provided "Resistant", “i "Intermediate" or "Susceptible", Bacteria Diameter of the zone of inhibition (mm) Sensitivity ay= Discussion: 1, What does narrow or broad spectrum antibiotic mean? 2, Were there any colonies found within the zones of inhibition, if found, Explain? 3. What-variable(s):could affect the results (diameter) in the Kirby ~ Bauer test? 4, What is the clinical significance of antibiotic sensitivity testing? 5. How:does the K ~B test differ from the MIC ? 6, Why is Mueller Hinton agar used rather than other media in K ~ B test? 8 |Exercise IX Lactobacillus activity in saliva Tooth decay has been linked to the normal flora bacteria in the oral cavity. Oral flora bacteria can form plaque and become embedded in lesions on the teeth, Through a combination of acid production by these bacteria and decomposition of tooth structure, dental caries form and increase in size. Bacteria growing in saliva and on the surface of teeth ferment available carbohydrates to produce lactic acid, Most of these bacteria are able.to ferment sucrose (table sugar) and complete its conversion to lactic acid in the plaque deposits on the teeth or in the saliva of the mouth: This fermentation reaction is completed before the sucrose containing food is comipletely chewed and swallowed. (those who have inflamed gums or teeth with dental caries will feel pain as. the acids produced by these bacteria irritate the lesions). The lowered: pH increases the solubility-of the enamel, resulting in dental. caries. The. formation of plaque and the lodging of food and bacteria in tooth lesions create anaerobic conditions that enhance the acid production through fermentation. When these organisms produce polymers of dextran, the lactic acid is held against the surface of the tooth as plaque, enabling their breakdown of tooth enamel and dentin. This results in more tooth decay. Sucrose is also important in the formation of the dextran layer. Method. 1. Rogosa SL agar. Rogosa SL agar is low pH and high acetate medium that suppresses most of the oral flora except the lactobacilli, (Is this medium selective ot differential?) In the Rogosa test, direct pour plate counts of the bacteria in saliva are made using Rogosa SL agar. This medium is highly selective for the genera Streptococcus (S) and Lactobacillus (L). the number of colonies that develop is assumed to be representative of the bacterial activity in the oral cavity. In the Rogosa test the actual number of acid producing bacteria in saliva is determined. The procedure assumes that there is some direct relationship between the numbers measured under laboratory conditions and the actual production of acid on the surfaces of the teeth. Number of bacteria / ml of salivafs “Susceptibility to dental caries | Approximate number / ml (Rogosa Test) Low <100/mi High <10.000/ mi Very high >10.000 7m ‘The value of the test lies in the relationship that exists between the production of acid by bacteria and the formation of dental caries. The accuracy and reliability of the test depends upon. correct sampling, accuracy in reading‘the test, and an.understanding of the limitations-of the :procedure;; Important: variables ‘include: the-timing of the sampling period (before or-after brushing -and flossing),.-eatly or Jate:in the day (food accumulation), andthe: nature’ of the.-diet:(high in. sucrose). Also, variation among individuals, especially. in:their susceptibility to.dental-caries, often influences test results. Laboratory objectives ‘This exercise will use the Rogosa test to determine susceptibility of individuals to dental caries, At the completion of this exercise, you will * Understand the relationship between fermentation and the production of acid in saliva and on the teeth; Understand the procedure used in this exercise to estimate bacterial count Understand the relationship between dietary sugar and the activity of streptococci and Jactobacilli in the oral cavity. Material needed for this lab. 1, Three tubes of SL Rogosa agar (9 mhl) foreach saliva sample being tested. 2. Sterile test tube 3. Sterile pipettes. 4. 3 sterile Petri plates, 50Laboratory procedures PIAA! 1, Obtain 3 tubes of Rogosa test agar. Melt the tube and hold at 50°C 2. Chew the paraffin block to stimulate production of saliva. Collect at least 2.0 ml of saliva in a sterile test tube ‘Transfer 1 ml of saliva into the first tube of Rogosa test agar. Mix thoroughly ‘Transfer 1 ml from the 1“ Rogosa tube into the second and mix thoroughly ‘Transfer 1 ml from the 2” Rogosa tube into the 3” and mix thoroughly Pour the tubes into sterile Petri plates. Let the agar solidify Incubate at 37°C record your results on the laboratory work sheet SLLab 9 Work Sheet What is the purpose of this exercise? 1, Bacterial count for saliva Dilution of saliva | Approximate number / ml Interpretation 1:10 1:100 1:1000 2. Complete the following chart using data from other groups in your laboratory section fo Student Number | Approximate number of | Number of fillings or cavities bacteria / ml 52Tan Questions : 1. Can you observe any correlation in the data collected? 2. Ifthe Students used a mouthwash prior to the sampling, how would you expect the results to change 53eT Exercise X Enzyme Linked Immuno Sorbent Assay (ELISA) ELISA stands for Enzyme Linked Immuno Sorbent Assay, one of the most widely used assays in the clinical laboratory. it can detect either antibody or antigen to the picogram (pg) range. It has replaced to a large extent the radioiminunoassay procedure because it does not require the use of dangerous radioisotopes and is easier to perform. It is one of the standard procedures used for the detection of antibodies to the HIV virus. The ELISA procedure:takes. advantage.of the:fact that :most-proteins will.bind firmly to the surface of several'differentkinds-of; plastic; wwsuelly: by! hydrophobic interactions between the non-polar residues of the protein:and the naturally ‘hydrophobic. components of the plastic. The -plastics. usually -employed-are “polystyrene or’ polyvinyl chloride, the most commonly used materials in.the manufacture of most micro titer plates. The test is perforined in four steps: 1, Adding the primary reagent (usually antigen or antibody) to the wells of the micro titer plate followed by, incubation then careful washing of the wells; 2, ‘adding the secondary reagent (osully an antibody specific forthe primary reagent) to the same wieils, followed by incubation and more washing; 3. adding the tertiary reagent (an antibody, specific for the secondary reagent, which has been conjugated by covalent bonding to an enzyme); 4, finally, the addition of a reagent to the wells which can be converted by the enzyme into acolored compound. ‘The wells containing the substance sought then assume a color that is directly proportional to the number of antibody molecules. bound: to. the entigen (primary reagent). Thus the ELISA procedure can be made quantitative or qualitative, according to methods used in the setup. Enzyme linked immunosorbent assay ELISA: test for tuman choroinie gonadotropin in serum or urine, You will be using the Beta- U- Test. A rapid and sensitive qualitative test for hCG in urine or serum. This test is a two sided sandwich ELISA which utilizes specific selected antibodies against hCG. 54Method 1 yawn Place one drop (50 ul) of the control, the references and the patient samples into correspondingly numbered wells Place one drop (50 ul) of antibody-enzyme-conjugate into each well ‘Mix gently and keep at room temperature for 5 minutes Wash the wells 5 times with tap water Add one drop (Saul) of Chromogen Reagent containing tetramethyl benzidine in methanol and substrate reagent containing hydrogen peroxide in buffered solution to each well Mix gently and keep at room temp. for 5-10 minutes Observe any color change and compare sample wells with positive and negative refergnces and control wells, Is your patient pregnant? 55.Appendix I The Traditional 4- Step Gram Stain 1 2, Prepare air dried, heat fixed thin bacterial smears. Step 1. Flood smears with 1% aqueous crystal violet reagent for Imin, briefly rinse in tep water, Dilute this solution if it is old or blue crystals are observed under oil - immersion in the finished Gram Stain, The erystal violet can be used even if highly diluted (up to 1:1000) without any effect on the reliability of the final reaction or timing in the procedure. * Step IL Flood crystal violet solution off the smears with excess Grams — iodine reagent for 1-2 min, briefly rinse. in tap water. Step M1. Decolorize very quickly in the very flammable mixture acetone: alcohol (I: 1) for 1-5 sec, the timing is very critical and is effected primarilly by the temperature of the laboratory and the decolizer itself, Generally this step is where the Gram Stain goes "wrong" usually by over docolorizing, resulting in gram - positive organisms that falsely appear gram - negative. Stop the decolorization reaction by plunging the slide into cold water, Step IV, Flood smears with the safranin counterstain reagent for about 2 min. For a more intens red color in gram - negative bacteria, some workers prefer 0.1 % aqueous basic fuchsin solution as a counterstain. Wash in water, dry completely, examine under oil with the oil immersion objective. The most reliable of "best" reacting area of the gram stained smear is usually at the periphery of the smear. 36 |EILE="F:\homework aaaaa.sav'. DATASET NAME DataSet] WINDOW=FRONT Page 1