VIRTUAL MICROBIOLOGY LAB GUIDE

Table of Contents:
General Introduction
     Objectives
     Expectations
Introduction to Diagnostic Laboratory Exercise
     Bacterial Morphology
     Diagnostic Microbiology
     The Gram Stain
     Streaking a Culture Plate for Isolated Colonies
Specific Instructions
     Part 1 - Gram-positive cocci
        Blood agar plates and hemolysis
        Catalase test
        PYR test
        CAMP test
        Bacitracin test
        Binax Strep A (point-of-care) test
        Optochin test
        Coagulase test
     Part 2 - Gram-negative rods
       MacConkey Agar Plate
       Citrate Slant

I. Objectives of the laboratory exercises

A. Provide familiarity with procedures used for culturing (growing) and identifying microorganisms of medical importance.
B. Aid in proficiency in submitting specimens for the identification of infectious disease in your future patients.
C. Provide clinical cases for diagnosis of infectious disease and its management.

These goals will be accomplished in the virtual microbiology lab exercise dealing with respiratory infection by identification of microorganisms in samples corresponding to case histories from patients. You will be asked to answer questions concerning the case histories and the general principles of diagnosis and of the etiological agents of respiratory diseases. For those who choose, practical experience can be obtained in streaking plates and making Gram stains in the limited wet lab.

II. What is expected of you

Your completion of the virtual lab giving you practical (decision making) and theoretical experience is important because:

1. You will be tested for your proficiency at reading and interpreting the results (note that the virtual exercise is sufficient, since your exam is electronic as well).
2. You will be tested on the theory behind the etiological agents and their identification.
3. You are expected to record the results of tests, identify the bacteria in the cultures, and arrive at diagnoses of the diseases.
4. You must submit your identifications and diagnoses along with completing an online (open note, open book) homework assignment.

INTRODUCTION TO DIAGNOSTIC LABORATORY EXERCISES

I. Bacterial morphology.

Bacteria are 100-1000 times smaller than most mammalian cells; they range from 0.4 to 3 microns (10^-3 mm) in diameter and several microns in length. To examine them you will need a light microscope with an oil immersion objective (100X). Microorganisms differ widely in shape and size. The majority of bacteria are either spheres (cocci) or rods (bacilli). A few occur as curved rods (vibrios) or in more complex shapes. Specific types of bacteria may also vary in size and grouping (single, clumps, pairs, chains, etc.). The shape and arrangement of the cells with their staining properties are used for classifying and preliminarily identifying clinical isolates. In response to a hostile environment, some bacteria adopt a dormant state by generating a spore, easily visualized with the light microscope.
 

II. Diagnostic Microbiology.
 

To identify the causative agent in infections, specimens are obtained, and each organism is isolated and identified. Microscopic examination of the specimen or the organisms is a first step. This can be an unstained ("wet mount") specimen or fixed specimen on a glass slide stained to visualize the microorganisms and other cellular elements. The most commonly used stain is the Gram stain, although other special stains (e.g., acid-fast stain) can be used to tentatively identify certain organisms.

To grow and isolate microorganisms, the specimen is spread ("streaked") onto agar media containing nutrients to yield colonies representative of each of the bacteria. Potentially important organisms are further tested for identification. A variety of methods is used - culture media which select for growth of groups of organisms (selective media), media containing indicators which cause different organisms to appear differently (differential media), biochemical tests, phage typing, antibody typing, and many others.
 

III. The Gram Stain.
 A. Introduction.

     The Gram stain is one of the most valuable and most generally used.  The Gram stain divides bacteria into two groups, the gram-positive organisms, which stain dark purple to black, and the gram-negative organisms, which take on the color of the counterstain, usually red. Bacteria are stained with crystal violet followed by Gram's iodine. These two solutions form a complex which, in gram-positive bacteria, is not washed away with ethanol; gram-negative bacteria rinse clear. To visualize the clear gram-negative bacteria, they are counterstained with a contrasting color. Red stains (e.g., safranin) are usually used.

     The ability of gram-positive bacteria to retain the crystal violet-iodine complex following treatment with ethanol varies with the age and species of bacteria and, to a lesser extent, the environment from which they were obtained. 
 

B. Procedure for Gram staining a specimen.


UF Students note - You need to understand the steps of the Gram stain and what happens at each step fr gram-positive and gram-negative bacteria, but you do not need to memorize the times.

 1. Using a sterile loop, transfer a loopful of tap water to a clean glass slide. Touch a loop to the desired colony, and mix the bacteria in the water on the slide. A VERY SMALL amount of bacteria will suffice.

2. Allow the specimens to dry on the slide at room temperature. Do not heat the slide to speed drying because this can distort the cellular morphology or staining properties of the organism. 

3. After the specimen has dried, heat-fix the slide. Gently heat the slide by passing quickly through the flame, specimen side up, 3-4 times. It should be warm but not hot to the touch.

4. Stain the bacterial smears by Gram's method as follows: Flood the slides sequentially with solutions a-d for the indicated times. 

(a) Crystal Violet--------------------------------1 minute

 Wash gently in tap water for 2-3 seconds.

 (b) Gram's Iodine (I₂-KI)-------------------------1 minute

 Wash gently in tap water, shake off excess water.
 (c) 95% alcohol-----------------------------------10 seconds 

Do not over-decolorize the specimen with alcohol. If you're going to screw up the Gram stain, this is the step! 

Wash gently in tap water, shake off excess water. 

(d) Safranin (counterstain)-----------------------20 seconds 

Wash in tap water and blot dry.

5. Examine with oil immersion optics (not at lower power). Move the condenser almost all the way up to touching the slide. Do not let the high/dry (40X) lens get into the oil. It will be very difficult to clean. 

6. Gram-positive organisms will be purple/blue. Gram-negative organisms will be pink to red. 

IV. Streaking a plate for isolation of colonies.
 A. Introduction.
     The single most important step in analyzing a specimen containing bacteria is to obtain isolated colonies of bacteria that arise from single cells. Attempts to identify bacteria in a clinical sample cannot be done unless isolated colonies are used. 

     To obtain well-isolated colonies, it is essential to disperse the inoculum (sample) on the surface of an enriched agar plate so that individual bacteria are well separated from each other. Ideally, each of the bacterial species present will produce a distinct colony type. The appropriate technique will be demonstrated by one of the instructors in each laboratory.

 B. Procedure.
1. With the loop, spread the inoculum back and forth across the upper 1/4 of the plate, keeping the lines of inoculation very close together (area 1 in this figure). Isolated colonies are not expected in this area. Do not use strong pressure, which will break the surface of the agar. Use the end of the loop, not its side when streaking.  Dispose of the loop in the biohazard bucket on the bench.

2. Turn plate approximately 90 degrees. Streak the plate across about 1/4 of the plate. (See area 2 of the figure.)  Dispose of the loop.

3. Turn the plate 90 degrees again, using the loop streak into the second area only a couple of times and then zig-zag across the remaining open area of the plate - being sure not to cross into areas 1 or 2 as this will put too many bacteria into this area that should hopefully contain isolated colonies.  Stab the first streak area a couple of times to accentuate hemolysis.

 4. Label plates and incubate inverted at 37 C.

Single colonies should appear in area 3.
 

Note: in drawings, lines should be closest together in Sec. 1 and progressively further apart in succeeding sections.

SPECIFIC INSTRUCTIONS

Part 1
Identification of Gram-positive Cocci

I. Introduction. 

The gram-positive cocci include organisms that are round and that usually occur in chains or pairs (streptococci) and those that occur in clusters or bunches (staphylococci). Infections by pathogenic gram-positive cocci are responsible for many bacterial diseases, ranging from superficial skin lesions to severe life-threatening infections. Other members of the group are fairly regular inhabitants of skin and mucous membranes, the so-called "normal flora."

     Blood agar plates.  The primary isolation from infectious material is usually made on sheep blood agar, a rich medium that supports the growth of many types of microorganisms. The appearance of colonies and red blood cell lysis are important diagnostic features. The most common streptococci and staphylococci can be divided into groups on the basis of their reactions on blood agar (examples are shown at labimage/imagky.html on the MMID home page): 

Alpha hemolytic - partial lysis of red blood cells, producing a greenish discoloration. The two most important groups are Streptococcus pneumoniae (pneumococcus), a frequent cause of lobar pneumonia and several other serious infectin, and the viridans group of streptococci, normal inhabitants of the oropharynx that may cause disease (e.g., endocarditis) when they invade the vascular system.  You will ALWAYS have viridans streps in a throat swab.

Beta hemolytic - complete lysis of red blood cells and clearing of the medium around the colony. Common pathogens which produce this reaction are Groups A, B, C and some D streptococci, as well as Staphylococcus aureus, the most common pathogenic staphylococcus.  For this lab, we are focusing on Group A strep (Streptococcus pyogenes) and Group B strep (S. agalactiae).
 

Gamma hemolytic - no apparent change in the medium (non-hemolytic is more descriptive). Staphylococcus epidermidis, a normal skin inhabitant. (See the insert of this figure to compare gamma and beta hemolysis.)
 

II. General Procedures. 

The purpose of this experiment is to make observations of some diagnostic features of the important streptococci and staphylococci. 

A. Differentiation of streptococci from staphylococci.
 

Although microscopic examination of stained smears presumptively permit distinction between these two groups of organisms, a definitive classification can be made on the basis of the presence or absence of the enzyme catalase. Staphylococci contain this enzyme, streptococci do not.
 
 Catalase test. Place a drop of 3% hydrogen peroxide on a clean microscope slide. Place a heavy loopful of cells from isolated colonies into the liquid (you may have to pick up four to five colonies if they are small). Immediate generation of gas bubbles constitutes a positive test. Avoid the inclusion of blood cells from blood agar plates as blood contains catalase. Lack of bubbles is a negative test. (Picture of results.)

                  catalase
2H₂O₂ --------------------> 2H₂O + O₂ (bubbles)

 B. Identification of group A Streptococcus (Streptococcus pyogenes).

1. PYR test. The PYR test has replaced the bacitracin test as the culture-based test for Group A streptococcus (S. pypogenes). It detects the presence of the enzyme Pyrrolidonyl Arylamidase. Pyrrolidonyl Arylamidase cleaves β-naphthylamide from a L-naphthylamide-β-naphthylamide substrate, and the released β-naphthylamide reacts with N, N-dimethylaminocinnamaldehyde present in a test strip yielding a red/pink color. The test is done by placing some colony material from a plate onto the test strip and looking for the color change. (Picture of results) In contrast, other beta-hemolytic streptococci, most notably Group B streptococcus (S. agalactiae), are PYR-negative.  The PYR test is done in conjunction with the CAMP test, since it yields complementary results.

2. CAMP test.  The CAMP test detects the presence of the CAMP factor (an acronym for the names of the discoverors), which is produced by some gram-positive cocci.  The CAMP factor potentiates hemolytic activity produced by Staphylococcus aureus.  Group B strep is CAMP+, whereas Group A strep is CAMP-.  The assay is performed by streaking S. aureus down the middle of a blood agar plate.  The unknown beta-hemolytic strep is cross-streaked across the staph, and the plate is incubated.  If the strep produces CAMP factor, an arrowhead-shaped zone of heightened hemolysis will appear at the junction of the streaks,  (Picture of results)  The CAMP test is done in conjunction with the PYR test.

3. Bacitracin test. The bacitracin test used to be the standard culture-based method for confirming S.pyogenes from other beta-hemolytic streptococci, such as Group B strep (S. agalactiae).  However, it has been replaced by the PYR and CAMP tests.  You should be familiar with the bacitracin test since it could show up on standardized exams.  Commercially available paper disks saturated with a solution containing Bacitracin will inhibit about 97% of all strains of Group A streptococci; other groups of beta-hemolytic streptococci will not be affected. Streak a blood agar plate with an isolated colony of beta-hemolytic streptococci (you're not looking for isolated colonies now). After inoculation, flame the provided forceps, and aseptically pick up a bacitracin disk (B or A disk). Place the disk on the plate and press gently onto the agar medium to ensure firm contact with the agar. Observe the plates for inhibition of growth(indicating sensitivity) after overnight incubation at 37. For Streptococcus pyogenes there will be a zone of inhibition of growth around the A disk. Note that the hemolysin might diffuse in the agar and make it look like the bacteria grew closer to the disk than reality. Also note that over inoculating the plate will make it difficult to interpret as well as under inoculating. 

4. BINAXNOW™ STREP A test. This is a point-of-care rapid test that is essentially the same as the at-home COVID-19 tests that we have all become familiar with as a result of the pandemic.  The test is detecting ANTIGEN using antibodies.  A swab is used to obtain a sample from the relevant site.  For Group A streptococcus pharyngitis, it would be the back of the throat.  For COVID-19, it is the nose.  The material in the swab is released with a buffer.  For this kit, the buffer is added to a well in the test card immediately before the test is run.  The swab is mixed with the buffer in the card.

Here's how the kit works (see the figure).  The buffer contains gold particles that are linked to the detection antibody, in this case anti-Group A carbohydrate.  If the sample has Group A carbohydrate, it will bind to the gold particles via the anti-Group A antibodies.  The solution migrates up the strip in the card via absorption.  The test line in the strip contains anti-Group A antibodies.  If the sample has Group A antigen, it will bind to the test line of anti-Group A antibody and capture the attached gold particles (the Group A carbohydrate is polymeric and can bind to more than one antibody).  This will form a red line on the test line.  This is necessary, but not sufficient, for a positive result.

Above the test line is a control line.  It has anti-IgG/IgM antibodies.  These antibodies will bind to the anti-Group A:gold particles and cause a red line, even if there is no Group A antigen in the sample.  Note that there will be plenty of anti-Group A:gold particles to enable this to happen, if it is a positive sample.  Failure of the red line to appear in the control line indicates a failure of the test.  This is most important if a negative result is obtained in the test line.  If the control does not turn positive, the negative test is meaningless.  Getting a positive test but negative control is unlikely, but would still invalidate the test.

This test is very specific because of the use of specific antibodies, and it is reasonably sensitive; however, it is possible that there is an infection that is low enough to not cause a positive result or that the sample was not adequate (poor swabbing).  Therefore, a negative test in suspected Group A strept pharyngitis should be followed with a more sensitive test such as culturing.  This is true for all point-of-care tests like this.

C. Differentiation of pneumococci from other alpha hemolytic streptococci.  

Optochin test. Pneumococci (but not other alpha-hemolytic streptococci) are inhibited by optochin. Apply a disk of filter paper containing optochin (O or P disk) to a heavily streaked plate (see procedure for applying the bacitracin disk). If the organism is a pneumococcus, a large zone free of bacterial growth will surround the paper disk (indicating sensitivity) after overnight incubation at 37C. If the organism is another alpha-hemolytic streptococcus, there will be no zone of inhibition around the disk, or at most a narrow one. Optochin is available commercially as "Taxos P" or "Optochin disk."
 

D. Differentiation of Staphylococcus aureus from non-pathogenic staphylococci.  

Coagulase test. The test which distinguishes S. aureus from non-pathogenic staphylococci is the coagulase test. Coagulase is a secreted enzyme of Staphylococcus aureus that causes plasma to clot (coagulate). The tube test is performed by inoculating an isolated colony into 10% rabbit plasma. After incubation at 37^oC for 2-4 hours, examine the tube for the presence of (picture). If the reaction is negative, incubate overnight and re-examine the tube.

 Part 2

Identification of Gram-negative enteric rods

I. Introduction.  

This part of the exercise will focus on the family Enterobacteriaceae, which includes several genera of medical importance. This is a large and diverse group, and the laboratory methodology for their identification has evolved over many years. You will work with Klebsiella pneumoniae and Escherichia coli.

The choice of medium for the initial isolation from a clinical specimen may depend on the specimen source. Usually specimens are cultured initially both on blood agar and a number of SELECTIVE media (e.g., MacConkey agar, which excludes the growth of gram-positive organisms because it contains bile salts). Media such as MacConkey also permit an assessment of the ability of the organisms to ferment lactose (DIFFERENTIAL media), which provides one of the key branch points in the diagnostic scheme.

These days systems are used which enable the simultaneous inoculation of many media and the evaluation of numerous biochemical characteristics. In this laboratory exercise, we will use the more classical (old fashioned) techniques because they form the foundation for all metabolic identification systems.

II. General Procedures

A. MacConkey agar plate. Streak samples for isolated colonies on MacConkey agar using same procedure as for Part 1. MacConkey agar is an example of a selective medium; it permits growth of gram-negative enterics but inhibits the growth of gram-positive bacteria. 

After 24 hours incubation, examine the plates to distinguish the two different colonial types. The MacConkey medium provides evidence as to whether each organism ferments lactose. Colonies which ferment lactose are red. This reaction is due to the action of acids produced by fermentation of lactose on the bile salts and the subsequent absorption of neutral red, a pH indicator, from the medium. Colonies of non-fermenters of lactose appear colorless. Example of results of a mixture of lactose-positive and negative bacteria on a MacConkey agar plate. 

E. coli and K. pneumoniae are usually lactose-positive. To make life interesting, you will also be given a lactose-negative mutant E. coli as part of this exercise. 

Also note that the E. coli and Klebsiella pneumoniae in your mixtures are vastly under represented. You will have a difficult time finding them on your blooad agar plates since they will be outnumbered by the staphs and streps. However, on the MacConkey plate, on which the streps and staphs don't grow, the gram-negative rods come forth in their true colors.

Some bacteria can use citrate as a source of carbon, while others cannot. Streak the surface of the citrate tube with an isolated colony from the MacConkey plate. Incubate the tube with the lid loose. Utilization of citrate as a carbon source results in a color change from green to blue. The indicator is bromthymol blue, which turns from green to blue at low pH. Read the test at 24 hour incubation at 37C. 

K. pneumoniae is citrate-positive, while E. coli is usually citrate-negative. Both of your E. coli strains are typical for citrate utilization.  In real life, as opposed to a contrived Virtual Lab, differentiating E. coli and Klebsiella involves an extensive battery of metabolic tests which is used to differentiate the gram-negative enteric rods..

OK, I've read through this stuff.  I'm an expert.  I'm ready to solve some cases!  (Click on the cases in the side bar.)