· web viewtests for the presence of the enzyme coagulase which, as you may expect, caused...

Unit 11: Microbial Blood Tests and Streak Isolation.By Patricia G. Wilber, Karen Bentz, and Heather Fitzgerald.Copyright Central New Mexico Community College, 2015

IntroductionCatabolism (breakdown) of blood and blood cells is an important characteristic used for the identification of microorganisms. When a specimen first comes into a medical diagnostic lab, such as Albuquerque’s Tricore, it is often inoculated on to three media plates: TSA-blood, Chocolate agar and MacConkey’s agar.

As you have seen in a previous lab, the MacConkey’s plate selects for Gram(-) organisms, making it a useful media to begin identifying unknown Gram(-) bacteria. However, none of the plates mentioned above select specifically for Gram(+) organisms, so Gram(+) identification depends on accurate results, keen powers of observation, and logic!

In this unit you will use media plates that contain sheep’s blood (TSA with blood, Chocolate agar), conduct the coagulase test, and review the Catalase test.

The TSA-blood plate media which contains defibrinated blood (fibrin, a clotting agent, is removed). It is non-selective and allows differentiation of bacteria based on alpha, beta and gamma hemolysis.

Chocolate agar plate contains partially hydrolyzed sheep’s blood and is non-selective. This is the only medium that supports the growth of Haemophilus species and is a good plate to use to observe colony morphology.

The Coagulase test tests for the presence of the enzyme coagulase which, as you may expect, caused coagulation of blood plasma. This test is useful for confirming the identification of Staphylococcus aureus.

Finally, you will review the catalase test and use the results to help characterize your bacteria, especially the Gram(+) species. In the small sample of species that we use in this lab, all the Gram(-) species are catalase positive, but keep in mind this is not true in the world at large! Anaerobic species are catalase negative and there are many Gram(-) anaerobes, such as organisms in the genera Bacteriodes and Fusobacterium (both of which are found in the gut).

TSA-bloodAs covered in Unit 7: Media: Supportive, Selective and Differential, TSA-blood is a non-selective media (most organisms grow on it) that allows differentiation of organisms based on blood hemolysis. Bacteria can hemolyze blood (blow up the blood cells) if they can produce hemolysins, enzymes that metabolize the protein hemoglobin. Different amounts of hemolysin production are described using the following terms: Beta, alpha and gamma. Of those, only beta produces true hemolysis. In alpha “hemolysis” hemoglobin is metabolized but the cells do

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B A

Figure created by Patricia G. Wilber, 2015.

not lyse. In gamma “hemolysis” no metabolism of hemoglobin occurs and there is no lysis of the blood cells.

Beta hemolysis: Highest production of hemolysins. Complete lysis of the red blood cells and complete metabolism of hemoglobin occurs resulting in a clear halo in the media underneath and/or spreading away from the bacterial growth.

Figure 11-1. Sometimes the clearing is very transparent (A), and sometimes there is a hazier appearance (B). This may be a result of different species or the age of the specimen. Transparency tends to increase over time.

Figure created by Patricia G. Wilber, 2015.

Alpha hemolysis: Less production of hemolysins. Partial digestion of the hemoglobin inside the red blood cells occurs, but the cells do not actually lyse. The hemoglobin is reduced to methemoglobin, which results in an olive greenish-brownish color in the media underneath the bacterial growth. It often appears that the bacterial colonies themselves are this greenish-brownish color.

Figure 11-2. Alpha hemolysis. An olive green to brownish color due to the reduction of hemoglobin to methemoglobin.

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Gamma hemolysis: No production of hemolysins. Growth of the bacteria occurs, but there is no lysis of the red blood cells or digestion of the hemoglobin in the cells by the bacteria. The bacterial growth is often a whitish color on the surface of the media.

Figure 11-3. Photograph of Alpha (α), Beta (β) and Gamma (γ) hemolysis

Accessed 7/29/2015 from https://www.studyblue.com/notes/note/n/block-5-study-guide-2013-14-hamill/deck/10254665, but licensed for use by the American Society for Microbiology, Creative Commons Attribution – Noncommercial – NoDerivatives 4.0 International license

Organisms that produce hemolysins are often opportunistic pathogens of humans. Many species produce more hemolysins under low oxygen conditions (like those found in damaged tissue), so blood plates are incubated in a candle jar which allows creation of a low oxygen environment.

Candle jars are large glass jars (like pickle jars). We place the incubated blood plates in the jar and place a candle on the top culture. The candle is lit, and the lid is screwed on the jar. Because of the oxidation (burning) of the candle, the oxygen in the jar is decreased to about 3% and the candle goes out.

The candle jar then has LOW oxygen, and the bacteria are stimulated to increase production of hemolysins. The condition in the jar is NOT anaerobic.

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What would happen to our cultures if we put them into an anaerobic environment?

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Chocolate agarChocolate agar is a supportive media that contains red blood cells that have been lysed in an autoclave. The lysed red blood cells release iron and amino acids as well as required bacterial growth factors such as NAD(factor V) and hemin(factor X). This supports the growth of the Gram(-), fastidious (picky) bacteria, Haemophilus haemolyticus, which require those nutrients but cannot access them when the blood cells are whole. Haemophilus haemolyticus does not produce hemolysins like the alpha and beta organisms we discussed above.

Haemophilus haemolyticus will ONLY grow on chocolate agar and is thus termed a “fastidious” (=picky) bacteria. Since the RBCs have been lysed to make their contents more readily available, bacteria do not need to produce hemolysins in order to grow well on the media, but sometimes they still will. You may see the alpha species turn greenish on the chocolate agar just like they did on the blood plate.

Chocolate agar, like TSA-blood, supports the growth of most common bacterial species.

Latex Coagulase TestThe latex coagulase test measures the ability of an organism to clot the plasma of the blood (the liquid portion) by using the enzyme coagulase. Ability to clot the plasma creates a faux capsule around the bacteria, making it difficult for host defense agents (like white blood cells) to perform phagocytosis. Production of coagulase by a bacterium is therefore considered an indicator of pathogenicity.

In a medical diagnostics lab (like Tricore), the latex coagulase test is used primarily to confirm identification of Staphylococcus aureus, after other tests have strongly suggested that the organism in question is, in fact, S. aureus. Therefore, coagulase is used near the end of a series of identification tests. Other pathogenic Staphylococcus species like S. saprophyticus may produce a positive reaction to the test, but the coagulase test is unlikely to be performed on this species because it would have been eliminated from consideration for this test prior to getting to that point.

Staphylococcus aureus is a Gram(+), catalase positive, coccus shaped, beta hemolyzer that can cause boils, carbuncles, impetigo, joint and bone infections, toxic shock syndrome and food poisoning. A closely related species, MRSA (Methicillin-resistant, Staphylococcus aureus), an highly antibiotic resistance form of S. aureus is responsible for over 11,000 deaths yearly in the US according to the Centers for Disease Control. Staphylococcus saprophyticus is Gram(+), catalase positive, and coccus-shaped, but is a gamma hemolyzer and is mainly found in urinary tract infections of women that are sexually active.

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Figure 11-6. The results of a coagulase test. A positive reaction is on the left (the organism CAN coagulate plasma resulting red clumps in the test) and a negative reaction (no ability to coagulate plasma; no clumping of any kind) is on the right.

The rabbit plasma coagulase test (which we are NOT using) does allow differentiation between S. aureus and S. saprophyticus, but the latex coagulase test does not. Therefore, it is important to follow a decision-making process prior to applying the latex coagulase test.

You will go through a decision making process and then perform the coagulase test on DAY 2 of this lab.

Catalase again.The catalase test allows us to determine if the organism produces the enzyme catalase. The catalase test is used diagnostically to separate Staphylococcus species (catalase +) from Streptococcus species (catalase -) and also separates Bacillus species (catalase +) from Clostridium species (catalase -).

The catalase test DOES NOT distinguish Gram(+) from Gram(-). The catalase test IS NOT an indicator of pathogenicity.

Blood cells, like all animal cells, contain catalase, so sometimes blood plates themselves will produce light bubbling if hydrogen peroxide is poured on them or if TSA-blood ends up in the bacteria tested. This is because the catalase in the blood breaks the hydrogen peroxide into water and oxygen (the bubbles).

You will perform the catalase test on Day 2 of this lab.

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B, 10 streaks through A

A, 1 cm smear of a tiny (very very tiny) bit of culture

C

D

E

DAY 1 Inoculation Materials:

1 TSA-blood plate per person 1 TSA-blood plate for two people 2 Chocolate Agar plates for two people Inoculating loops Incinerator (on) Sharpies Candle jars Cultures:

o Streptococcus pyogenes (Spy) (Gram(+), Beta)o Staphylococcus aureus (Sa) (Gram (+), Beta)o Serratia marcescens (Sema) (Gram(-), AlphaBeta)o Staphylococcus saprophyticus (Ss) (Gram(+), Gamma)o Proteus vulgaris (Pv) (Gram(-), Gray Gamma Beta)o Haemophilus haemolyticus (Hh) (Gram(-), fastidious)

Procedure:I. TSA with Blood

A. Streak isolation of a gamma organism (Ss).1. Each person will perform this procedure. 2. Properly label the bottom of your TSA-blood plate.3. Use Ss4. Sterilize your loop.5. Using aseptic technique, perform a streak isolation (Unit 2 for review).

6. Re-sterilize your loop and return to the metal canister7. Place your plate in the candle jar for incubation.

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Gram (-) AlphaBeta

(Sema )Gram (+) Beta

(Sa)

Gram (+) Beta (Spy)

B. Hemolysis. 1. Work with a partner and use one TSA-blood plate for both of you.2. Divide the blood plate into thirds by drawing on the outside of the agar side of the

plate with the Sharpie.3. Using a sterilized loop, inoculate the plate with species and fishtail/squiggle pattern

shown in Figure 11-4 below.4. Be sure to label each section so you know what is growing where!5. Re-sterilize between inoculations.6. Re-sterilize and return the loop to the metal canister at the end.7. Place the inoculated plate (agar side up, like normal) in the candle jar for incubation.

Figure 11-5. Inoculation of the TSA-blood plate.

Image created by Patricia G. Wilber

Precautions Incubate the blood plates in the candle jar to maximize hemolysin production (and thus

hemolysis). Haemophilus haemolyticus (Gram(-), fastidious) will not grow on simple TSA-blood. DO

NOT USE this organism in Part I!

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II. Chocolate AgarProcedure:

1. Work with a partner.2. Divide your two chocolate agar plates into thirds by drawing on the outside of the agar

side of the plate with a Sharpie. (see Figure 11-5 below)3. Using a sterilized loop, inoculate all six species on the plates, using the inoculation

fishtail/squiggle shown in Figure 11-5. 4. Re-sterilize the loop after each use!5. Be sure to label each third so you know what species is where.6. Place the inoculated plates in the rack for incubation.7. Re-sterilize and return the loop to the metal canister.

Figure 11-5. Inoculate all six species on your two chocolate agar plates in the pattern shown. Label each third so you know what you put where!

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Day 2.

Catalase Test https://www.youtube.com/watch?v=mtK91MOd650 Latex Coagulase Test https://www.youtube.com/watch?v=qtzSuKfW0Zg

Video created by Corrie Andries and Karen Bentz

Materials: Your TSA-blood and chocolate agar plates with the various species growing. The species you should have grown:

o Streptococcus pyogenes (Spy) (Gram(+), Beta)o Staphylococcus aureus (Sa) (Gram (+), Beta)o Serratia marcescens (Sema) (Gram(-), AlphaBeta)o Staphylococcus saprophyticus (Ss) (Gram(+), Gamma)o Proteus vulgaris (Pv) (Gram(-), Alpha Beta)o Haemophilus haemolyticus (Hh) (Gram(-), fastidious)

Coagulase test kit (2 per table) Hydrogen peroxide bottles (2 per table) Wooden dowels

Results and Interpretation.Obtain your cultures, observe the results, do some procedures and answer the questions.

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Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links:

https://www.youtube.com/watch?v=qtzSuKfW0Zg

I. TSA-blood In order to observe any type of hemolysis (alpha, beta or gamma), the culture must first grow. Our blood plates are not selective, so most species will grow!

A. Streak Isolation1. Observe your streak isolation, photograph it and place the photo in the space below.

Did your streak isolation grow? (It was supposed to). Yes or no?______.

a. If it grew, great!b. If it did not grow, why might that be?

2. What type of hemolysis did you expect to see on your streak isolation plate? ______________.

Why did you expect that?

3. What type of hemolysis did you see on your streak isolation plate?? _____________________.

4. How do you know you have the type of hemolysis you wrote for 3?

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5. If your hemolysis type is gamma, . If your hemolysis type is not gamma why might this be?

6. Are hemolysins (enzymes that break down hemoglobin) produced by gamma hemolyzers? Explain.

7. Did you achieve isolation on your plate? (8-10 isolated colonies in streak area 3 or 4).a. If so, YAY!b. If not, how can improve/ modify your technique to get better results?

8. Look at your isolated colonies. Notice the size, appearance and color of the colonies.

B. Blood hemolysis: beta and alpha results.1. Observe your other blood plate and photograph the results. Insert the photo here.

2. Did each third show growth?___________ (They were all supposed to have growth). If there was no growth, indicate on the photo where the no growth occurred and hypothesize as to why in the space below.

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3. On the same photograph, label the areas with alpha and beta hemolysis. DEFEND each label.

II. Chocolate Agar.1. Photograph your results and paste them here. You should see growth in all six areas.

Note: This is the ONLY medium that supports the growth of Haemophilus haemolyticus.

2. What unique characteristics do you observe about the growth of each species? (color, appearance, opacity, feathering away from the streak, etc. There are probably no individual colonies so you cannot observe colony morphology!) Record these observations in Table 11-1. You DO need to be able to see that the species are different from each other. Recognition of growth differences can help begin the process of species identification (and you might need to apply this in your final project!)

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Table 11-1. Differential Descriptions of the Bacterial Growth you ObservedBacterial species Unique Characteristics of Growth you Observed

3. When looking at your results on the chocolate agar, can you see any evidence of hemolysin production for any of the species you tested? (hint: green) If no, leave the table blank. List the species and the evidence.

Species Evidence of hemolysin production

III. Latex Coagulase TestProcedure:

1. Obtain your blood plate with three species on it,2. Observe the hemolysis patterns.3. Only beta hemolytic species should be considered as candidates for the coagulase test.

Identify all the areas on your blood plates that show beta hemolysis. List the species that you grew on the blood plate that are beta hemolytic below. (There may be two or three)

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4. Only beta hemolytic, Gram(+), coccus-shaped species should be considered for the coagulase test. You grew more than one beta hemolytic species,

Do a Gram Stain to establish which are Gram(+) and coccus shaped. Work with other groups at your table. You will have two or three species to Gram stain. Each person can test ONE species and you can compare and consult so that you can:

List the beta hemolytic, Gram(+), coccus-shaped species:

In addition, calculate cell size (as a review for the midterm). The Field Diameter at TM=1000X is __________. (See your Unit 2—you calculated the Field

Diameter.)The number of cells that fit across the Field Diameter is _________________.

The formula is FD/# of cells that fit across. The cell size is __________________.

5. Only beta hemolytic, Gram(+), coccus-shaped, catalase + species should be considered for the coagulase test. Perform a catalase test on the beta hemolytic, Gram(+) coccus-shaped species you have listed above. USE A SAMPLE OF THE SPECIES YOU HAVE LISTED FROM YOUR CHOCOLATE AGAR PLATE.

Mini catalase test review: Put a little puddle of hydrogen peroxide on a slide; get a dowel, push end of the dowel gently onto the bacterial growth you want to test to get some bacteria (and no agar) on the end of the dowel; put end of dowel with bacteria on it into puddle of hydrogen peroxide; DO NOT STIR; watch for immediate production of bubbles (=positive).

List the beta hemolytic, Gram(+), coccus-shaped, catalase + organism(s)

6. You will test the one species that is beta hemolytic, Gram(+), coccus-shaped and catalase positive with the latex coagulase test.Name this species: ____________________________________Check with your instructor to verify the species you are planning to test is the right one.

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7. Do the Coagulase Test! (Directions below.)

The Latex Coagulase Test1. Obtain a test kit.2. Get a test card with one circle on it. 3. Shake the reagent bottle.4. Put ONE drop of the test reagent (it is red) on the card.5. Use the Chocolate agar plate and also use a plastic stick provided in the test kit. Pick up

a good amount of bacteria OF THE SPECIES YOU DETERMINED YOU SHOULD TEST (the Gram(+), coccus-shaped, Beta hemolytic, catalase positive organism)from your CHOCOLATE AGAR PLATE but (NO AGAR) and put it on the test paper next to the drop of the reagent.

6. Stir them together in slow circles with the plastic stick. Stir slowly until your see clumps.7. Record or photograph the results.

Results:Figure 11-7. Positive (red clumps are visible) and negative (no clumping) results of the

coagulase test.

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Figure 11-8. Results of your coagulase test. If your results were positive, record them in the positive column. You should not have negative results if you followed the protocol correctly.

Positive

Interpretation.1. What species did you use for the latex coagulase test?

2. Based on the results, did the species you tested with the latex coagulase test produce a positive or a negative result? Defend your answer!

3. Based on your results is the species you tested a possible pathogen? Defend your answer!

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4. Based on your results, what enzyme does your organism possess?IV. Catalase again.The catalase test is a very commonly performed test. Just to review it, do the following.

1. Obtain your blood and chocolate agar plates. 2. Test each PLATE (the media itself) and all 6 species to see if catalase is present.3. Do this test LAST because you will put hydrogen peroxide directly on the plates as you

do not need the plates after this procedure. Plus, this is a lot faster than doing the dowel technique.

4. Put a few drops of hydrogen peroxide on the TSA-blood plate. DO NOT let the hydrogen peroxide come in contact with any of the bacteria growing on your plate. Observe closely. Record your results in the table provided.

5. Put a few drops of hydrogen peroxide on the chocolate agar plate, away from the bacterial growth. Observe closely. Record your results in the table provided.

6. Put a few drops of hydrogen peroxide on each of the six species you grew. Do this one species at a time. Observe closely. Record your results in the table provided.

Table 11-2.Item tested Look up and

record whether the organism

tested is Gram(+)

or Gram(-)

Indicate whether the catalase result was

positive (write POSITIVE) or negative

(write NEGATIVE)

TSA-blood plate NA Was the plate + or -?

Chocolate agar plate NA Was the plate + or -?

Serratia marcescens

Staphylococcus aureus

Streptococcus pyogenes

Proteus vulgaris

Staphylococcus saprophyticus

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Haemophilus haemolyticus

Compare your results to the Results Key (see online webpage or the end of Unit 13) to see if you interpreted your results correctly.

1. Based on your results, does the Catalase test distinguish Gram(+) from Gram(-)? Explain your answer.

2. Staphylococcus species should be catalase ___________ and Streptococcus species should be catalase ________________. Do your results support this? (yes or no).

3. If an organism bubbles when hydrogen peroxide is added, what enzyme does it produce?_______________.

4. Does producing catalase make an organism pathogenic? _________

5. If an organism is catalase positive , it might be an ob_________ ae____________, or it might be a Fa___________ an________________.

6. If an organisms is catalase negative, it MIGHT fall into these four aerotolerance categories.

Mi_________________________ Cap_______________________

Aero_________________ ______________

Ob_______________ an______________

7. All obligate aerobes should produce catalase. True or False.

8. Organisms grown in our lab are never obligate anaerobes. True or False. Defend this answer.

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Post lab questions

1. Based on your results on the TSA blood plate, which of the species that you tested produced hemolysins? How do you know this?

Species name Evidence of hemolysin production

2. Haemophilus haemolyticus is fastidious. What does this mean?

3. Chocolate agar contains RBCs that were lysed when heated in an autoclave. How does this aid bacterial growth?

4. List the four characteristics a species should meet in order to be considered for the coagulase test.

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5. Based on your results for each species you tested, determine which enzymes each has and whether or not there is evidence for pathogenicity.

Species Hemolysins (Write type of

hemolysis—“alpha”, “beta” or “gamma”

followed by “has

hemolysins” or “does not

have hemolysins”)

Coagulase(Write “has coagulase”

or “does not have

coagulase”)

Catalase(Write “has

catalase” or “does not have

catalase”)

Evidence for pathogenicity?Write “no evidence” or list the

enzymes that indicate pathogenicity

Serratia marcescens

Staphylococcus aureus

Streptococcus pygoenes

Proteus vulgaris

Enterococcus faecalis

Haemophilus haemolyticus

The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Alex Silage for IT support, and our dean, John Cornish.

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