LABORATORY 2

Basic Bacteriologic Techniques I. OBJECTIVES

1. To master viewing bacterial specimens with the microscope. Practice using oil immersion to focus on specimens.

2. To become familiar with routine bacteriologic procedures: sterile transfer,

inoculation of media, and slide preparation. 3. To utilize the streak-plate method for isolation of individual bacterial colonies

from pure and mixed broth cultures.

4. To gain familiarity with the Gram stain procedure.

4. To perform antibiotic sensitivity testing using different microorganisms.

6. To prepare an acid-fast stain.

7. To observe demonstrations of bacterial stains routinely used in clinical microbiology laboratories for organism identification.

II. BACKGROUND All of the bacteriology laboratories in this course will rely heavily on the successful use of the light microscope and oil immersion lens to examine the morphology and staining characteristics of bacteria. The skills developed in this laboratory section should be carried with you during your clinical years and will be useful in the care of your future patients. The ability to stain and identify bacteria in clinical specimens such as sputum, CSF, and wound cultures will benefit your patients and enhance your performance as a medical student and as a resident on wards. Therefore, focus your attention on learning the basic bacteriologic methods presented during laboratory exercises. Bacteria are everywhere! On the bench tops, in water, soil and food, on your skin, in your ears, nose, throat, and intestinal tract (normal flora). The diversity of bacteria present in our environment and on and in our bodies is incredible. When trying to study bacteria from the environment, one quickly discovers that bacteria usually exist in mixed populations. It is only in very rare situations that bacteria occur as a single species. However, to be able to study the biochemical, morphological, and physiological characteristics of an individual species, it is essential that the organism be separated from the other species that are normally found in its habitat. In other words, we must establish what is called a pure culture of the microorganism. A pure culture is defined as a population containing only a single species or strain of bacteria. Contamination means that more than one species is present in a culture that is supposed to be pure. Contamination does not imply that the contaminating organism is harmful, it simply means that the contaminating organism is unwanted in the culture that you are trying to isolate and study. Special techniques, commonly referred to as aseptic pure culture techniques, must be used to obtain a single isolated strain for study.

Historically, bacteria have been the cause of some of the most deadly diseases and widespread epidemics of human civilization. A tremendous amount of time and resources were used to develop methods of treatment for infectious diseases. Antibiotics have dramatically changed the prognosis of patients exhibiting symptoms of bacterial infection. Vaccines were developed that are able to prevent the spread of infectious disease in populations. Advances in technology and molecular medicine (automation, polymerase chain reaction) have added greatly to the clinical microbiology diagnostic armamentarium. However, clinical microbiology remains an interpretive science in which an individual’s experience and judgment are pivotal. It pays to understand the biology of bacterial pathogens and how they are able to produce disease.

Some infectious diseases are distinctive enough to be identified clinically. However, most infectious agents can cause a wide spectrum of clinical symptoms in humans. Conversely, a patient may present with symptoms that may be attributed to infection with any one of many pathogens. For example, Influenza virus infection causes a wide variety of respiratory syndromes that cannot be distinguished clinically from those caused by streptococci, mycoplasmas, or more than 100 other viruses.

Therefore, identification of a specific infectious pathogen typically requires the clinical microbiology laboratory. The clinical microbiology laboratory will test patient specimens for microorganisms that are, or may be, a cause of the symptoms experienced and will provide information (when appropriate) about the in vitro activity of antimicrobial drugs against the microorganisms identified.

Importance of the clinical laboratory. Specimens are collected from a symptomatic patient and submitted to the laboratory for characterization and identification. The diagnosis is determined by the use of many types of evaluation: microscopic, culture, serology, and genetic. Additionally, it is helpful for the clinician to know which antibiotics the pathogen is susceptible to (http://gsbs.utmb.edu/microbook/ch010.htm).

A. OPERATION OF THE MICROSCOPE

Note that there are three objectives: a low power (10X), medium or high dry power (40X), and oil immersion objective (100X). The oil immersion objective is always used for the morphological study of bacteria. The three objectives may be distinguished by the difference in focal length or numerical aperture, as indicated in the following table:

Common Designation

Focal Length Numerical Aperture

Lens Magnification

Eye Piece Magnification

Final Magnification

Low Power 16.0 mm 0.25 10X 10X 100X High Dry 4.0 mm 0.65 43X 10X 430X Oil Immersion 1.0 mm 1.25 (in oil) 97X 10X 970X

http://www.hendrix.edu/homes/fac/sutherlandM/CellWeb/Techniques/microparts.html The following directions should be followed routinely when operating the microscope: 1. Rotate the low power objective (yellow stripe) into place and put the slide on the stage. 2. Move the low power objective down as close as possible from the slide using the

coarse control and adjust upwards until the specimen is in focus as seen through the ocular. Find the specimen first with the 10X objective.

3. Light adjustments may be necessary. It is possible to place the light source in focus

by raising and lowering the condenser. Center the light source. 4. Adjust the condenser and iris diaphragm to a position giving the strongest even light. 5. Focus on the specimen with the coarse adjustment finishing with the fine adjustment.

6. Once in focus, you are now ready to use the oil immersion lens. Move from the yellow stripe, to the red stripe, and finally to the white stripe objective. To focus the oil immersion lens:

a. Select an appropriate field. b. Open the iris wide. c. Turn the 40x objective away enough to place a drop of oil on the specimen/

slide. Place a small drop of immersion oil on the specimen. d. Swing the oil immersion lens into the oil. e. Use only the fine focus to bring the object into focus. Never use the coarse

focus when the oil objective is in place. The slide could break and the objective could be damaged.

f. At the end of the class period, clean off the objective with lens paper. 7. Again, note that the oil immersion lens must be used for examining bacteria. Do not

attempt to identify bacteria under lower power; you will be seeing only artifacts.

Wipe the upper lens of the eyepieces with clean lens paper both before and after use. The oil should always be wiped from the oil immersion lens with lens paper prior to putting away the microscope. Microscopes should be clean, with the low power objective in place when put away.

B. ROUTINE BACTERIOLOGIC PROCEDURES: STERILE TRANSFER, INOCULATION OF MEDIA, AND SLIDE PREPARATION

Sterile transfer of bacteria and inoculation of media

Since diagnostic bacteriology is concerned essentially with the isolation and identification of single species of bacteria, techniques must be acquired and employed assiduously in order to ensure that contamination does not occur. Sterile transfer refers to the techniques used to move bacteria from single colonies or from a pure culture source to another culture media or test tube.

With any type of microbiology technique (i.e. working with and culturing bacteria), it is important not to introduce contaminating bacteria into the experiment. Because contaminating bacteria are ubiquitous and are found on fingertips, bench tops, etc., it is important to avoid these contaminating surfaces. When working with a microorganism, the round circle at the end of the inoculating loop and the surface of the agar plate should not be touched or placed onto contaminating surfaces. Proper sterile technique will also help ensure safe microbiological practice. Use the following guidelines for all transfers of bacteria during this laboratory course.

1. The inoculating loop and needle generally are used for making transfers. The same methods apply to both.

Source: http://www.antonides.nl/en/entogen.htm

2. To transfer a culture, flame the loop until it glows. Allow the loop to cool

sufficiently so that the organisms picked up by the loop will not be killed (10-15 seconds).

3. After the transfer is completed, sterilize the loop carefully. To avoid splattering

infectious material, first dry and gradually burn the material in the inner (cooler) zone of the flame.

4. Flame briefly the mouth of test tubes, flasks, etc. both before and after each

transfer. Learn how to remove and replace screw caps or metal caps with the same hand that holds the loop. (Your TA will demonstrate this elegant technique.)

5. Work while seated, avoid unnecessary movement and confine manipulations to the vicinity of the flame.

6. Practice and attention to detail will allow you to work both rapidly and

accurately. (If you get an opportunity, observe Jan West’s masterful technique.)

C. TYPES OF CULTURE MEDIA A satisfactory culture medium must contain sources of carbohydrate, nitrogen, inorganic salts and, in certain cases, vitamins or other growth factors. These may be supplied by meat infusions, beef extract, and peptone. Certain bacteria require the addition of other substances, such as serum and blood. Carbohydrates may be desirable at times. Dyes may be added as indicators of metabolic activity or because of their selective inhibitory powers.

1. Broths (liquid) – maintain growth of most organisms

2. Agar (solid) – culture plates, slant or stab tubes

3. Kinds of media:

• General purpose – agar or broth. Supports growth of most organisms, e.g.,

nutrient agar and trypticase soy agar • Enriched media - Certain organisms require growth- promoting ingredients

such as blood, glucose, serum, egg, etc. The media containing ingredients which enhance their growth-promoting qualities are enriched media, e.g. blood agar, chocolate agar and Loeffler medium.

• Enrichment media - 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 unhampered and 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.

• Differential - usually agar. Differential media have chemical constituents which characterize different bacteria by their special colonial appearances in the culture e.g. MacConkey agar contains lactose as a 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 turn red. The red lactose fermenting colonies can be differentiated from the pale non-lactose fermenting colonies.

Examples of agar plates used for culturing bacteria. Source: http://www.netpath.net/~billbsr/Page26.htm

Frau Hesse and the Use of Agar in Microbiology

Fanny Angelina Eilshemius was born in 1850 in New York to a wealthy Dutch immigrant family. As a young woman, she toured Europe. While in Europe, she met and married a German doctor, Walther Hesse (1874). Dr. Hesse joined Robert Koch's laboratory in 1881 to study the new science of microbiology. Frau Hesse, nicknamed Lina, was a talented artist and drew illustrations for her husband's publications. At that time, gelatin was used as a solidifying agent for microbiological media. Gelatin plates frequently melted on hot days and many of the bacterial isolates digested (liquified) the gelatin. According to Wolfgang Hesse (ASM News Vol 58#8 p. 425-428, 1992), a descendant of Dr. and Frau Hesse, Dr. Hesse asked his wife why her jellies and puddings stayed solid in warm weather when his microbiological media did not. She told him that she used agar-agar as a solidifying agent when she made jellies in hot weather. She had learned of agar-agar as a youngster in New York from a Dutch neighbor who had immigrated from Java. Dr. Hesse subsequently tried agar as a solidifying agent in bacteriological media and found that it worked wonderfully. Since then, agar has been the standard solidifying agent for microbiology.

D. SLIDE PREPARATION In order to examine the bacteria in mixed or pure culture under the light microscope, it is necessary to fix them to a glass slide and stain them. 1. Cleanliness of Slides. Microscope slides must be clean and free from grease

so that satisfactory films may be obtained. A clear slide will permit a drop of water to spread into a thin film.

2. Division of Slides. For convenience, several films may be prepared and stained

on one side. Use a glass-marking pencil to divide the slide into sections on the side opposite to that on which the films are to be made.

3. Cultures in Fluid Media. Transfer one or more loopfuls of the material to a slide to form a film about a centimeter in diameter. Deposit and spread the material gently to minimize disruption of cellular arrangements.

4. Cultures on Solid Media. Transfer one or more loopfuls of tap water to the slide. Using the loop or the inoculating needle (the latter is preferred), transfer a small portion of the bacterial colony or culture fluid next to the drop of water. You should not see lumps of material on the slide. Mix the two and spread gently to form a thin film.

5. Fixation. Place slid on dryer until it is completely dry. This fixes the organisms to the slide and kills them.

Practice is required to prepare films that are suitable for staining and microscopic examination (see below). A satisfactorily stained preparation will allow examination of individual cells and of cellular arrangements. The usual error involves films which are too thick, so that only clumps and masses of material are seen and cellular morphology is obscured. Keep in mind that a single loopful of a heavy broth culture will contain 106 -107 organisms.

E. ISOLATION OF MICROBIAL SPECIES IN PURE CULTURE Bacteria are rarely found as pure cultures in nature. Many clinical samples submitted for analysis to the microbiology laboratory are mixtures of organisms. Within these mixtures may reside both pathogenic (disease-causing) and non-pathogenic bacteria. An organism must first be isolated and grown in pure culture before it can be studied accurately. Plate cultures offer a means of isolating pure species of organisms in a simple manner. The following two methods are generally used: 1) the streak-plate method, and 2) the selective inhibition method. Streak-plate Method -- This method is used most commonly. a. The inoculum is transferred to an area near the periphery of the plate.

b. The inoculum is spread back and forth over the surface of about one-fourth of the plate.

c. The plate is now turned 90°.

d. Sterilize the loop in the flame, allow it to cool, and touch it to the last streak on

the original inoculum. e. With this diluted inoculum, streak the second quadrant of the plate and, without

flaming the loop, continue streaking the third and then the fourth quadrant of the plate (this procedure will be demonstrated).

f. Invert the plate and incubate.

Isolated colonies of bacteria will appear on the surface of the agar after incubation. Each colony usually represents the growth from a single organism. A pure culture may be obtained from a well-isolated colony by transferring a portion with a wire needle to an appropriate culture medium.

F. SEPARATION OF ORGANISMS FROM MIXED CULTURES

Separation of different bacteria in a mixed population is accomplished by spreading an appropriate sample of the mixed culture on the surface of a solid agar medium in such a way that the individual bacteria are well separated from one another. If the medium is nutritionally adequate, the isolated bacterial cells will multiply in a fixed position to form a clone composed of the progeny from a single organism. The clone or colony produced is usually of sufficient size to be visible.

When a particular organism is sought, it is often possible to use a medium or conditions which will support the growth of the organism in question and at the same time prevent the growth of undesired bacteria by exclusion of required nutrients or by addition of selective inhibitors or both. This principle will be illustrated in this laboratory exercise through the use of the following media:

1. Blood agar plates (BAP), a general medium.

2. Columbia CAN Agar (CNA), contains colistin and nalidixic acid, which inhibit DNA replication of Gram-negative organisms.

3. MacConkey agar (Mac), a selective and differential medium. MacConkey agar is selective in that it inhibits the growth of Gram-positive organisms due to the presence of bile salts and crystal violet. It is differential in that Gram-negative bacilli that ferment lactose (coliform bacilli) produce red colonies and non-lactose fermenters produce colorless colonies. The basis for this differentiation is that when coliforms ferment lactose (the only carbohydrate in the medium), acid is produced in the area of the colony. The acid precipitates the bile salts and allows absorption of neutral red (pH indicator in medium), which turns red to purple at acid pH.

4. Mycosel medium contains cycloheximide and chloramphenicol, which inhibit

protein synthesis in prokaryotic cells. Growth of non-pathogenic fungi and bacteria are inhibited. These inhibitors do not penetrate pathogenic fungi and yeast cell walls.

G. GRAM STAIN

Of the staining techniques routinely employed in diagnostic microbiology, the Gram stain is most widely used. The principle of this stain has remained unchanged since the procedure was devised in 1884 by Dr. Christian Gram. The technique, however, has been modified over the years in keeping with improvements in the qualities of dyes. Bacteria either retain the primary stain crystal violet (which has a bluish-purple color) in this procedure, and are designated Gram-positive, or they retain only the second dye, usually safranin (reddish pink) and are designated as Gram-negative.

Briefly, Gram-positive organisms have cell walls whose permeability properties are changed by exposure to alcohol or acetone, so that the crystal violet-iodine complex is not removed by subsequent washings with this solvent for short periods of time. In the case of Gram-negative bacteria, the crystal violet, in combination with iodine, stains the cell, but because of the nature of the cell wall, the alcohol or acetone treatment removes the stain. The colorless cells are then stained with a dye of a different color. Because of changes that occur in the walls of bacteria as a result of aging and death, old or dead Gram-positive cells may stain Gram-negative. This is especially true of certain species of Gram-positive bacteria and may occur in clinical specimens from patients who have been given antibiotic therapy.

Gram + Cell Wall Gram - Cell Wall

Gram Stain Procedure Reagents: Primary Stain: Crystal violet solution Mordant: Iodine and potassium iodine solution Decolorizer: 95% ethyl alcohol and acetone, 1:1 Counter Stain: Safranin O solution Method: Paine and Thomas (1963). Gram staining without the clock.

New England Journal of Medicine 269: 941. a. Do not look at the clock.

b. Apply crystal violet to the heat-fixed smear. Allow stain to remain on the slide only as long as it takes to replace the bottle of stain. Immediately wash the slide with tap water.

c. Flood the slide with Gram's iodine solution, leaving the iodine on the

slide only as long as it takes to replace the bottle. Immediately wash the slide with tap water.

d. Apply 4 or 5 drops of decolorizer (alcohol or acetone) and immediately

wash the slide with tap water. Apply the decolorizer carefully and do not try to decolorize the thick parts of the smear. The thin areas of the smear, which are the only areas that can be examined satisfactorily, will then be over-decolorized.

e. Apply safranin, leaving the stain on the slide only as long as it takes to

replace the bottle, and immediately wash the slide with tap water. Blot the slide dry (do not wipe!) and examine microscopically.

Although the Gram stain is easy to perform, several factors influence the results, e.g. thickness of the film, amount of decolorization, and the age of the culture employed. Constant practice is required in order to obtain consistent results. This is the most important skill you will master in this course. Take the time to learn it well.

Gram + Gram –

Steps in Gram Staining:

PURPLE PINK

(http://www.spjc.edu/hec/vettech/vtde/ATE2639LGS/Gramstain.htm)

Gram Stain and General Microscopy Troubleshooting Guide

Problem Probable Cause Corrective Action

Slide is almost focused, but is hazy or unclear

Inadequate or no immersion oil

There should be enough oil to make a seal between the slide and the 100X objective

Slide is clear under 10X but cannot be focused under oil Slide is upside down Check slide and flip over if necessary

Organisms appear to have a halo around them or cannot be focused

Oil was added before slide was dry

Make a new slide. Allow slide to dry longer or blot more carefully before adding oil

Slide is in focus, then a shadow appears

Air bubble trapped by lens Rotate objective back and forth

Floating material is seen Objective scraped the smear

Clean objective; gently remove oil from slide and refocus more carefully

Nothing is visible on the slide Smear too thin Remake with a thicker smear Slide wiped to dry it Blot slides; never wipe

Smear was not heat fixed Repeat; heat fix the smear

Excessive deposits of stain crystals on slide

Inadequate rinsing between steps

Repeat; wash off one stain completely before adding the next

Gram positive organisms have odd angular shapes

Viewing crystal violet crystals

Repeat: Do not allow dye to dry on the slide. Do not allow crystal violet to remain on slide longer than suggested times

Gram negative organisms look more like needles than rods

Viewing safranin crystals

Specimen appears mixed with large oval cocci when it should be one organism

Yeast growing in the staining reagents

Repeat after the instructor has filtered the reagents

Gram positive cells appear Gram negative Overdecolorization Decrease decolorizartion time

Non-viable organisms Use a fresher culture Gram negative organism appears Gram positive Underdecolorization Increase decolorization time

Mixture of Gram positive and Gram negative organisms from a pure culture

Smear is too thick Remake smear

H. ANTIBIOTIC SENSITIVITY TESTING

The simplest and most widely used method for antibiotic susceptibility testing is the antibiotic disc agar diffusion procedure. Sterile filter paper discs impregnated with specific concentrations of various anti-microbial agents are placed on an agar plate which has been inoculated with the culture to be tested. After overnight incubation, zones of growth inhibition appear around the disc. The size of the inhibition zones depends upon:

1. Diffusibility of the antibiotic 2. Test conditions 3. Inherent susceptibility of the test strain

Under very specific standard conditions the size of the zone can be used as a measurement of the susceptibility of the strain. The Kirby-Bauer standard method is based on the comparison of zone sizes with quantitative dilution tests as well as a correlation with achievable drug levels for each antibiotic (Bauer, Kirby, Sherris, and Turk [1996], Am. J. Clin. Path. 45: 493).

I. ACID FAST STAIN

Acid fast cells contain mycolic acid (a saturated fatty acid) in the cell walls. Mycobacteria and certain species of Actinomycetes resist staining with ordinary dyes. Moreover, these organisms, once stained, retain the dye with great tenacity, so that treatment with alcoholic mineral acid fails to remove the dye. Non-acid-fast bacteria are readily decolorized by the acid-alcohol. Kinyoun's method eliminates the use of heat, which is required by the Ziehl-Neelsen technique. You will use Kinyoun method as given below: Reagents

Basic fuchsin 4.0 gm Phenol crystals 8.0 gm Alcohol, 95% 20.0 ml Distilled water 100.0 ml

Method

a. Prepare the smear in the usual manner. b. Flood with basic fuchsin and allow to sit undisturbed for 5 minutes; rinse with water. c. Flood with acid alcohol and allow to sit undisturbed for 2 minutes; rinse with water. d. Apply the methylene blue counterstain to the slide and allow to sit for 1 minute; rinse with water. e. Air dry (DO NOT BLOT) and examine under microscope.

Acid-fast bacteria will appear as bright red organisms against a blue background.

This is an acid fast stain of Mycobacterium tuberculosis (MTB). Note the red rods— hence the terminology for MTB in histologic sections or smears: acid fast bacilli.

Microscopically, Mycobacterium avium intracellulare infection is marked by numerous acid fast organisms growing within macrophages. Lots of bright red rods are seen, particularly in macrophages, in this acid fast stain of lymph node.

J. DEMONSTRATIONS OF SPECIFIC STAINS AND MICROSCOPES

There are a number of specific stains that are useful in a clinical microbiology laboratory in order to identify specific structures of a microorganism. Various bacterial structures may be visualized in the light microscope with the use of specific staining techniques. Certain of these structures are of importance in the identification of pathogens.

1. CAPSULE STAIN

Many microorganisms secrete a highly viscous gum-like material which adheres to the cell wall. In some instances, as in the Pneumococci, the presence of a capsule is associated with virulence. The chemical structure of the capsule is often used as a basis for classification within a bacterial species.

2. SPORE STAIN Spores are formed primarily by species of Bacillus or Clostridium and survive

harsh environments, remaining dormant for many years, until conditions for growth become more favorable. Spores are able to withstand periods of desiccation, high temperature, UV light, toxic chemicals, and a lack of suitable nutrients. Because a spore’s outer coat is an effective barrier to chemicals, spores generally stain poorly. However, the use of very hot dyes apparently allows the coat to expand and allow the dye to permeate.

3. FLAGELLA STAIN

Flagella are long filamentous appendages on organisms classified as non-fermenters. They are too thin to be seen by ordinary microscopy unless heavily coated with special stains containing a precipitating agent such as tannic acid, which make the flagella thicker and more visible.

4. CELL WALL STAIN

The bacterial cell wall is a fairly rigid structure that preserves the integrity of the protoplast. It plays an important role in metabolism and cell multiplication. In this stain, the normally negative bacterial cell surface is made positive by treating it with a cationic surface agent. The positive cell surface will now bind a negatively charged chromophore of an acidic dye. The cell wall stain indicates the presence of cell walls, which is important in the identification of bacterial species that lack a cell wall (mycoplasmas).

5. METHYLENE BLUE STAIN

Useful in the identification of bacteria such as Corynebacteria that contain inclusion bodies, also called metachomatic granules. Bacteria will stain a light blue, and metachomatic granules, if present within the bacterial cell, will stain a deeper blue.

6. DARKFIELD

Darkfield microscopy is used to examine living microorganisms that are mostly invisible with a brightfield microscope. It uses a special condenser and reflected light so the specimen appears bright against a black background. This type of microscopy is particularly useful for primary syphilis identification in exudate.

III. PROCEDURE

Day 1 Step 1 Teaching assistants will demonstrate and answer your questions regarding: a. Standard operation of the microscope. b. Sterilization of an inoculating loop with an open flame.

c. Transfer of cultures from tube to plate and how to streak plates for isolation of colonies.

Step 2 Isolate colonies from pure cultures of microorganisms (Work independently)

Pure cultures of representative microorganisms in broth and agar will be provided: Escherichia coli (labeled E. coli), Staphylococcus aureus (labeled S. aur), Candida albicans (labeled C. alb).

a. Streak the E. coli, S. aureus, and C. albicans for isolation on a

individual blood agar plates (BAP). Use a different plate for each organism! Invert labeled plates and place in containers at the end of the tables for overnight incubation at 37 C.

b. Prepare a Gram stain from each of the three organisms from the pure

broth culture. Follow the Gram Stain protocol on page 30 and record your results on page 37.

Candida albicans Staphylococcus aureus Budding structures (arrows) Gram (+) cocci in clusters

Escherichia coli Gram (-) bacilli

Colony Morphology on BAP/Size, Color

Gram Stain Gm + = purple Gm - = pink

Microscopic Morphology

1. E. coli

2. S. aureus

3. C. albicans

Step 3 Gram stain each of the three organisms growing on the trypticase agar plates

(Work independently) Step 4 Isolate individual colonies from a mixed culture of microorganisms

(Work independently)

A mixed culture consisting of Escherichia coli, Staphylococcus aureus, and Candida albicans will be provided. Four types of media will be supplied: Blood agar, Coumbia CNA agar (CNA), MacConkey agar, and Mycosel agar.

http://www.indstate.edu/thcme/micro/basic.html

a. Streak the mixed culture on the different media plates supplied using the

streak dilution method and incubate at 37°C for 18 hours. b. Make a smear of the mixture and stain according to the Gram Stain protocol.

Identify the different morphologies and their staining characteristics. Step 5 Antibiotic Sensitivity Testing (work in pairs; 1 large plate per two students)

Dilutions (1:100) of broth culture of various organisms will be provided for completion of the antibiotic sensitivity testing.

a. Using a sterile cotton swab, inoculate a large agar plate with the diluted

culture. Streak the surface evenly and completely in three directions to

provide a solid lawn of growth. You should make sure the surface of the agar is completely covered with the organism.

b. Place antibiotic discs on the agar with an automatic disc dispenser. Note:

Be sure to use the appropriate set of discs for Gram-positive and Gram-negative organisms. Seat the discs firmly into the agar with forceps that have been flamed and cooled. Be careful not to scar the agar. It will not require force to seat the discs into the agar. Label plates. Leave at the table to be picked up and incubated overnight.

Day 2

NOTE: It is essential that you master the Gram stain and streak-plate isolation methods. They are necessary in order to identify unknowns that will be coming up in future laboratory sessions. If you are still having trouble staining the microorganisms, ask your TA for help and keep trying.

Step 1 Verify that isolated colonies were obtained on plates streaked from cultures.

• Record notes about the colonies. Are they large, small, pinpoint? • Do they have a particular color? Do they produce an odor? • Did they grow on some plates, but failed to grow on others? • Are they dull or shiny in appearance? • Are they raised on the plate or are they flat?

Step 2 Use an isolated colony to perform a Gram stain for each organism.

• Record whether the organism is Gram positive or Gram negative. • What shape are the microorganisms? • Do they form clusters, or are they spread out across the field?

Step 3 Evaluate organisms growing on the different media plates by performing a

Gram stain for each.

• Record observations about the colonies growing on the different types of media on page 40. Also, complete the chart on p. 40 regarding selective and differential media.

Step 4 Interpret the antibiotic sensitivity plates

• Measure the diameter (in mm) of the clear zone surrounding the antibiotic disc.

• The chart on page 41 will provide the information necessary to determine whether the organism is resistant, intermediate, or sensitive to the antibiotic.

• Record your results on pages 42 and 43.

S. aureus exhibits different sensitivities to antibiotics. http://www2.austin.cc.tx.us/microbugz/48antibio.html

Step 5 Perform an Acid Fast Stain on the sample provided

a. A slide from a clinical specimen will be provided. Perform acid-fast staining by the Kinyoun method (as described on page 33).

b. Examine your slide for the presence of acid-fast organisms, and compare your results with others at your table.

Step 6 Demonstration of specific stains.

Demonstration slides will be set up on microscopes in the center of the laboratory. Observe each staining technique and record your findings in the table on page 44.

Selective Media Media: Contains: Inhibits:

Selects for: Differentiates

between: Mechanisms of differentiation:

1. PEA

2. MacConkey

3. Mycosel

INTERPRETATION OF DISK SUSCEPTIBILITY TESTING

Measure the diameter of the clear zone surrounding the antibiotic disc to the nearest mm. The following chart will provide necessary information as to whether it is sensitive or resistant.

Antibiotic Disc Potency Resistant Intermediate Sensitive Amikacin (AN) 30 μg 14 15-16 17 Amoxacillin/Clavulanic Acid (AMCL) 30 μg 19 ------- 20 or more Ampicillin (AM) Gram-negative and and Enterococci 10 μg 11 or less 12-13 14 or more Staphylococci 20 or less 21-28 29 or more Others 11 or less 12-21 22 or more Cefotaxime (CTX) 30 μg 14 15-22 23 Cefotetan (CTT) 30 μg 12 13-15 16 or more Ceftazidime (CAZ) 30 μg 14 15-17 18 Cephalothin (CF) 30 μg 14 or less 15-17 18 or more Clindamycin (CC) 2 μg 14 15-20 21 Erythromycin (E) 15 μg 13 23 Gentamicin (GM) 10 μg 12 or less 13-14 15 or more Imipenem (IPM) 13 14-15 16 Levofloxacin (LVX) 5 μg 13 14-16 17 Minocycline (MI) 30 μg 14 15-18 19 Oxacillin (OX) 10 11-12 13 Penicillin-G (P) Staphylococci 10 μg 20 or less 21-28 29 or more Other organisms 10 μg 11 or less 12-21 22 or more Piperacillin/Tazobactam (PIP) 17 μg 17 18-20 21 Sulfa/Trimeth (SXT) 30 μg 12 or less 13-16 17 or more Tobramycin (NN) 10 μg 12 15 Vancomycin (VA) 30 μg 8 or less 9-11 12 or more

ANTIBIOTIC SENSITIVITY RESULTS

Gram-Positive Organisms Sensitive = S Intermediate = I Resistant = R

ORGANISM Antibiotic S. aureus S. epidermidis Enterococcus

AMCL

CF

CC

LVX

OX

MI

P

SXT

VA

ANTIBIOTIC SENSITIVITY RESULTS

Gram-Negative Organisms

Sensitive = S Intermediate = I Resistant = R

ORGANISM Antibiotic Pseudomonas E. coli Klebsiella

AN

AM

CTX

CTT

CAZ

GM

IPM

LVX

SXT

PIP

NN

Record your observations of the demonstrations here (use drawings) Staining Technique

Microscopic Appearance

Organism Demonstrated

1. Capsule Stain

2. Spore Stain

3. Flagella Stain

4. Cell Wall Stain

5. Methylene Blue Stain

6. Darkfield

7. Phase contrast

QUESTIONS YOU SHOULD BE ABLE TO ANSWER: 1. Why is the Gram stain said to be a differential stain? 2. What are the differences between a Gram-positive and a Gram-negative cell wall? 3. Can a Gram-positive cell ever stain Gram-negative? 4. What are some different stains that can be used to help identify an organism? 5. How might a physician use antibiotic sensitivity when treating a patient? 6. Why should one not lay an inoculating loop on the table? 7. Why is it important to use aseptic transfer techniques in a clinical microbiology

laboratory?

“Typhoid Mary’s Gallstones To Blame” American Society for Microbiology General Meeting, Florida, May 2001.

Nearly 100 years ago at least 10 outbreaks of typhoid fever in New York City were traced to the apparently health cook, Mary Mallon. Typhoid Mary, as she later became known, may have been an efficient reservoir for the disease because her gallstones were coated with typhoid bacteria, researchers told this week’s annual meeting of the American Society for Microbiology in Orlando, Florida. Thanks to improved sanitation and antibiotics, typhoid fever, which is caused by the bacterium Salmonella typhi, is no longer common in the developed world. Where it does occur, it causes fever and sometimes death if untreated. Between 3 and 5% of those infected show no signs of illness and become carriers of the disease. The reason for their amazing ability to harbor and pass on the bacteria, sometimes for years, had until now been a mystery. One thing carriers often have in common is gallstones. So microbiologist Angela Prouty took sterile gallstones from patients at the University of Texas Health Science Center in San Antonio, where she works, and infected them with S. typhi. The bacteria rapidly formed tough biofilms over the surface of the gallstones. Bacteria in a biofilm are tightly bound to one another and to the surface they coat. Tenacious biofilms of S. typhi on gallstones could explain why some people harbor the disease so well, says Prouty. “It’s a very good way for the bacteria to keep put and not get washed away,” she says. Bile and gallstones were essential to induce S. typhi to form biofilms. Say Prouty: “We tried it with pebbles and it didn’t work.” The bacteria’s predilection for the gallbladder makes good evolutionary sense. Bile aids digestion by draining from the gallbladder into the lower intestine. ”The bacterium can shed itself back into the environment,” say Prouty. Now Prouty needs to demonstrate the presence of S. typhi in biofilms of the gallstones of infected patients, says Salmonella microbiologist Brad Cookson of the University of Washington in Seattle. “But it’s a step towards understanding the interaction and its significance,” he says. Even if there had been antibiotics back in Typhoid Mary’s day they would have been of little use. Most antibiotics do not get to the gallbladder, and those that do can rarely break of biofilms. “The only way to get rid of the bacteria is to get rid of the gallbladder,” Prouty says.

http://www.nature.com/nsu/010524/010524-12.html