Lab Notes for Exam 2 Section Ex. 3-8: Acid-Fast Stain




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Lab Notes for Exam 2 Section
Ex. 3-8: Acid-Fast Stain

The acid fast stain distinguishes bacteria in the genus Mycobacterium, which are associated with the diseases tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The presence of unique waxes, mycolic acid, in the cell wall make it possible to differentially stain these organisms for diagnostic purposes. Simple stains do not readily penetrate these waxy cell walls, and heat must be used to melt the waxy substance. Once stain penetrates the melted waxy cell wall, these organisms are difficult to decolorize, even when acid is added to the decolorizer. Acid readily disrupts the ionic bond formed between simple stains and cell walls composed of peptidoglycan when simple staining or Gram staining. Cells that retain the primary stain in the presence of acid are referred to as acid fast. Non-acid fast cell walls decolorize with the acid alcohol decolorizer and must be stained with the counterstain, methylene blue, to be visible. Cells that are acid-fast appear cherry red or magenta in color, while non-acid fast bacteria will appear blue.



Organisms:

Slant culture of Mycobacterium smegmatis

• Slant culture of Staphylococcus aureus

Materials:

• Carbol Fuchsin, Acid Alcohol Decolorizer (NOT Gram stain decolorizer), Methylene Blue

• Hot plate and beaker setup in fume hood

Procedure:


  1. Prepare two slides of a mixed specimen containing both Mycobacterium and Staphylococcus aureus, using the same procedure you used to prepare the mixed specimen during smear preparation. Remember to add a small drop of water to the slide to suspend the cells. Use the loop to break up any clumped cells and to spread out the cells evenly. If you fail to spread out the cells you will have large masses of cells and will not see individual cells.

  2. Heat-fix your preparation by placing the slides on a warm heating block until specimen is completely dry.

  3. Place one slide over a beaker of hot water (just below the boiling point) on a hot plate. Cover the slide with a small piece of bibulous paper. Remember, the second slide is a backup. Do not use it unless your first attempt is unsuccessful.

  4. Flood the slide with carbol fuchsin and allow the stain to steam for 5 mins. Add more stain as it begins to dry along the edges. Make sure that you do not let the stain dry out and handle the hot slide and hot plate carefully.

  5. After 5 minutes carefully remove the slide with a clothes pin (it will be hot!), cool it for a minute or two, then gently wash it with water.

  6. Decolorize with acid alcohol for a few seconds. This is the critical step in this procedure. Add the acid-fast decolorizer drop by drop. Do NOT use the 95% ethanol Gram stain decolorizer.

  7. Wash with water again. One or two more decolorizing and washing steps may be necessary. There may still be some residual stain left even after repeated washing and decolorizing.

  8. Counterstain with Methylene Blue for 1 - 2 minutes.

  9. Wash with water, blot dry, and examine under the microscope.

Examine the slide(s) you have prepared. Which organism is the acid-fast organism? What color is it? Which organism is not acid fast? What color is it?
Ex. 3-10: EndoSpore Stain

Purpose: Perform the spore stain and identify bacterial endospores and vegetative cells

The spore stain is a differential stain used to distinguish between metabolically active vegetative cells and dormant, resistant spores. Spores are formed by some bacteria when their nutrient supply is exhausted. Like other differential stains such as the Gram Stain and Acid-Fast Stain, the spore stain requires the use of two contrasting stains, a primary stain followed by a counterstain of a different color.



Organism: An agar slant culture of Bacillus cereus

Materials:

  • Malachite green stain and safranin stain

  • Hot plate and beaker setup in fume hood

Procedure:

  1. Prepare two slides according to the procedure for a smear from a solid medium. The second slide is a back up.

  2. Heat-fix your preparation by placing the slides on a warm heating block until specimen is completely dry.

  3. Place one slide over a beaker of hot water (just below the boiling point) on a hot plate. Cover the slide with a small piece of bibulous paper. Remember, the second slide is a backup. Do not use it unless your first attempt is unsuccessful.

  4. Flood the slide with malachite green and allow the stain to steam for 5 mins. Add more stain as it begins to dry along the edges. Make sure that you do not let the stain dry out and handle the hot slide and hot plate carefully.

  5. After 5 minutes carefully remove the slide with a clothes pin (it will be hot!), cool it for a minute or two, then gently wash it with water.

  6. Apply the safranin counterstain for 1-2 minute.

  7. Wash the safranin off gently with water.

  8. Blot with bibulous paper and examine. The spores will appear green and the vegetative cells will appear orange-red.

NOTE:

Make certain that your microscope is put away correctly. The lowest power objective should be in position over the stage opening, the slide should be removed, no oil should be present on the stage or on any of the dry lenses, and the microscope should be placed into the correct box. NEVER USE KIMWIPES, PAPER TOWELS OR KLEENEX TO CLEAN THE LENSES - only the lens paper in your lab kit.



Nutritional Requirements: Differential and Selective Media

Purpose: To become familiar with various types of commonly used selective and differential media and their uses.

Ex. 4-4: Mannitol Salts Agar

Organisms: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus; E. coli

Media: One MSA (mannitol salts agar) plate per pair

Procedure:

1. Divide plates into four sections. Label each section with the name of a different organism.

2. Using aseptic technique, do a straight line inoculation, with a loop, of each organism into the appropriate section of the plate. Incubate the plate, upside down, at 37° C.

3. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth using a 0-3 scale. Growth on the surface indicates salt tolerance. Note any changes in the color of the agar itself. A yellow color indicates the ability to ferment the sugar mannitol.



Ex. 4-5: MacConkey Agar

Organisms: Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, and Proteus vulgaris

Media: One MacConkey agar plate per pair

Procedure:

1. Divide plate into four sections. Follow same procedure as for Ex. 4-4. Incubate the plate at 37° C.

2. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth using a 0-3 scale. Growth indicates that the organism is Gram negative. Note the color of the growth and the surrounding agar. A bright pink/magenta color indicates lactose fermentation. A bright pink "cloud" in the agar indicates Escherichia coli, produced by the large degree of lactose fermentation.



Ex. 5-3: phenol red broth

Purpose: test of the capacity of bacteria to oxidize various carbohydrates via a fermentative pathway

Organisms: E. coli, Alcaligenes faecalis, Proteus vulgaris, & S. aureus

Media: Four tubes of each of the following, per pair of students: phenol red glucose (dextrose) broth, phenol red lactose broth, phenol red sucrose broth

Procedure:

1. These tubes contain an additional glass tube, called a Durham tube, inside the medium. Before inoculating, invert any tubes that may have bubbles to remove the bubbles.

2. Label the tubes and inoculate each tube with the appropriate organism.

3. Incubate tubes at 37° C. Make sure the tops are loosened by 1/2 turn when they are incubated.

4. Next lab period: Observe the results and look for a color change (yellow, orange, pink, deeper red), a bubble in the Durham tube (large, medium, small or barely visible), and turbidity (cloudy or not).
Catalase and Oxidase Tests

After the Gram reaction and cell shape has been determined, the catalase test and the oxidase test are often the first tests performed to identify an unknown organism.

If an organism is Gram negative rod, then a negative oxidase test indicates the organism is a Enteric bacteria; a positive oxidase test indicates a non-enteric organism. We will be using the oxidase to test to determine which rapid ID test should be used to identify the unknown organism you isolated from hamburger.

If an organism is a Gram positive coccus, a negative catalase test indicates it is a Streptococcus; a positive catalase test indicates is a Staphylococcus. The coagulase test can then be used to further identify the species of the Staphylcoccus.


Ex. 5-5: Catalase Test


This test is used to indicate whether a microorganism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. This enzyme is important to aerobic organisms because it detoxifies hydrogen peroxide. Hydrogen peroxide forms during aerobic metabolism when components of the respiratory chain donate electrons to molecular oxygen. The other enzyme produced by many microorganisms to detoxify hydrogen peroxide is peroxidase, however, no oxygen is evolved from the breakdown of hydrogen peroxide by peroxidase. This test is used to differentiate between Streptococcus and Staphylococcus species.

Organisms: S. aureus, S. epidermidis, Streptococcus salivarius and any other organisms used in today’s exercises. Use only cultures grown on agar for the catalase test.

Materials Needed: Microscope slides, hydrogen peroxide in small bottles

Procedure: Place a drop of hydrogen peroxide on a microscope slide, add a loopful of the organism and observe for immediate bubbling. Catalase positive organisms will exhibit bubbling, catalase negative will not.

Ex. 5-6: Oxidase Test

This test detects the presence of the enzyme, cytochrome oxidase, which transfers electrons from cytochrome c to molecular oxygen in the electron transport chain. Many aerobic bacteria, such as Neisseria sp. and Pseudomonas sp., have cytochrome oxidase. On the other hand, many facultative anaerobes such as those in the family Enterobacteriaceae (Gram negative facultative anaerobes), are oxidase negative because they lack cytochrome c in their electron transport chain, and therefore, do not have cytochrome oxidase. This test is often used to differentiate between enteric (facultative anaerobic) and non-enteric (aerobic) gram negative bacteria.



Organisms: E. coli, Pseudomonas aeruginosa and Alcaligenes faecalis grown on TSA plates

Materials Needed: Oxistrips (Tetramethyl-p-phenylenediamine dihydrochloride)

Plastic sterile disposable transfer loops



Procedure: Place a Oxistrip on a paper towel. Using a PLASTIC loop, smear the test strip (the part with a matte appearance) with a loopful of inoculum. A positive result will produce a dark purple color within 20 to 30 seconds. No color means the organism is negative for cytochrome oxidase.
EXOENZYMES

The following tests detect the presence of exoenzymes. Exoenzymes are enzymes that are secreted into the surrounding medium and work on substrates found outside the cell. In general, these exoenzymes are hydrolytic and break down large biomolecules that are too large to be easily transported into the cell. These biomolecules must be broken down into their smaller building blocks before they can be made available as a nutrient source for the cell. Starch must broken down into glucose, protein into amino acids, and triglycerides into fatty acids and glycerol.


Ex. 5-12: Starch Hydrolysis Test – This is a test for the amylase exoenzyme that breaks down starch into glucose

Organisms: E. coli and B. cereus

Media: One starch agar plate per pair

Procedure:

  1. Divide plate in half and label each section with the appropriate organism. Do a straight line inoculation of each organism onto the plate. Incubate, inverted, at 37° C.

  2. After incubation, flood the plate with Gram’s iodine. Observe for the presence of dark color surrounding the inoculation line. Dark color up to the edge of the inoculum indicates the absence of starch hydrolysis. A clear zone surrounding the inoculum indicates a positive reaction, meaning that the starch in the medium has been broken down (hydrolyzed). Discard the plate after it has been reacted with iodine.

Ex. 5-14: Casease Test This is a test for the casease exoenzyme that breakdown the milk protein, casein, into amino acids

Organisms: E. coli, S. marcescens, and Pseudomonas aeruginosa

Media: One Casein agar plate

Procedure:

  1. Divide the plate into thirds and label each section of the plate with one of the three organisms. Do a straight line inoculation into the correct section of the plate. Incubate, inverted, for 48-72 hours at 37° C.

  2. Observe for a clear zone surrounding the inoculum which is indicative of casein hydrolysis.

Ex. 5-15: Gelatinase Test – This is a test for the presence of the gelatinase exoenzyme, which breaks down the protein, gelatin, to amino acids

Organisms: E. coli, S. marcescens, and Pseudomonas aeruginosa

Media: Three gelatin deeps

Procedure:

  1. With a needle, inoculate the deeps in a straight line stab inoculation. Incubate the tubes for at least 48 hrs. at 37° C.

  2. After incubation, chill the tubes in an ice bath or the refrigerator and look for the presence or absence of liquefaction after chilling. Remember that any color change you observe is due to the pigment produced by that organism, not by any by product of the gelatinase reaction. If the reaction appears to be negative, return the tubes to the 37° C incubator. For some species, liquefaction can take up to a week to observe.


Ex. 5-17: Lipase Test for lipase exoenzymes that breakdown lipids to glycerol and fatty acids

Organisms: E. coli, S. marcescens, and Pseudomonas aeruginosa

Media: Tributyrin (lipid) agar plate

Procedure:

  1. Divide plate into thirds. Do a straight line inoculation of each organism into the appropriate section of the plate. Incubate, inverted, for at least 48 hours at 37° C.

  2. A clearing of the agar around the inoculum indicates the breakdown of lipids in the medium. Lipid hydrolysis may take longer for some lipid + species. If you observe incomplete clearing around an inoculum, return your plate to the 37° C incubator and check it again next period.


DIFFERENTIATION OF GRAM NEGATIVE RODS

The following tests can be used to differentiate Gram negative rods. In particular, the Indole test, Methyl Red test, Voges-Proskauer test, and Citrate utilization are sometimes known collectively as the IMViC test and are used whenever preliminary tests indicate an unknown organism belongs to the family Enterobacteriaceae. The Enterobacteriaceae include pathogens such as Salmonella and Shigella, occasional pathogens such as Proteus and Klebsiella, and normal intestinal flora such as Escherichia and Enterobacter. These tests are not entirely inclusive and they may be used for other purposes as well. The table below summarizes the characteristics of a few common species.





Laboratory Tests for Differentiation of Gram Negative Rods

Organism

Enteric/Nonenteric (E/N)

Carbohydrate Fermentation

H2S Production

Indole Production

MR Reaction

VP Reaction

Citrate Utilization

NO3 Reduction

Urease Activity

Oxidase Activity

Gelatin Liquification

Starch Hydrolysis

Lipid Hydrolysis

 

 

Glucose

Lactose

Sucrose

 

 




 

 

 

 

 

 

 

 

Enterobacter aerogenes

E

AG

AG

AG+/–







+

+

+











Escherichia coli

E

AG

AG

A+/–



+

+





+











Proteus vulgaris

E

AG



AG+/–

+

+

+



+/–

+

+



+





Salmonella arizoniae

E

AG+/–



A+/–

+



+



+

+











Alcaligenes faecalis

N















+/–





+







Pseudomonas aeruginosa

N















+

+



+

+



+

A = acid

G = gas


+/– = result can vary between strains of the same species
The tests presented in Chapter 5 of the Lab Manual are all differential. Differential tests are based on the reaction of an indicator reagent with a biochemical product that is produced in the presence of a specific enzyme made by an organism. The differential media must contain the appropriate substrate for the reaction as well as the indicator. It is up to the organism to make the product., which then reacts with the indicator. Selective media are used to enrich for a particular type of organism if you are working with a mixed culture specimen that may have, for instance, been collected from a patient. Remember that some media are both selective and differential, while others are selective only or differential only.
Ex. 5-4: Methyl Red and Voges Proskauer Test - The methyl red test is used to identify bacteria that produce mixed acids (lactic, acetic, or formic) as a result of glucose fermentation. The acid end products lower the pH of the growth medium to 5.0 or lower. The Voges Proskauer test identifies bacteria that form non-acidic end products as a result of glucose fermentation. The final fermentation end products of these bacteria are ethanol and acetylmethylcarbinol (acetoin). These tests are frequently used to distinguish between Escherichia coli and Enterobacter aerogenes.

Organisms: E. coli and E. aerogenes - broth cultures

Media: Two tubes of MRVP broth

Procedure:

  1. Inoculate organisms into the appropriate tubes. Incubate at 37° C.

  2. After incubation, aseptically transfer 1 ml of the culture with a sterile bulb pipette to one of the sterile tubes in the hood. You will now have two tubes for each organism. The tube with 4 ml of culture will be used for the methyl red test. The other tube with the l ml of culture will be used for the Voges Proskauer test. Since you have two organisms, you will have a total of four tubes.

  3. To the tubes for the Methyl Red test, add 5 drops of the methyl red indicator. When methyl red is added, the indicator turns the medium red in the presence of acid. This is done under the hood. Read the tubes immediately and look for color change from pale yellow to red. Red color indicates the presence of acidic end products from glucose fermentation.

  4. To the tubes for the Voges Proskauer test, add 10 drops of Barritt’s solution A (5% alpha naphthol) and then add 10 drops of Barritt’s solution B (40% potassium hydroxide). Allow 15 to 20 minutes to pass and observe for the appearance of a red ring at the top of the culture. This color change is indicative of a positive result. The absence of a red ring is a negative result for the presence of acetylmethylcarbinol.

Note: All chemicals are to be used under the hood and with extreme care!
Ex. 5-7: Nitrate Reduction Test - Some microorganisms are capable of using inorganic molecules other than oxygen as the terminal electron acceptor during an energy yielding metabolic pathway such as respiration. When this occurs, the process is called anaerobic respiration. Carbonate, sulfate, and nitrate are examples of inorganic terminal electron acceptors other than oxygen. The process whereby nitrate is reduced to nitrite, ammonia, or molecular nitrogen, is known as nitrate reduction.

Organisms: E. coli, P. aeruginosa, and Alcaligenes faecalis

Media: Three tubes of nitrate broth per pair

Procedure:

  1. Inoculate and incubate tubes at 37° C.

  2. After the specified incubation time, add reagents A and B (IN THE HOOD) as indicated on the reagent bottles and make observations. Interpretation of the results can be confusing. Read the manual carefully!

    1. If nitrate is reduced to nitrite, the nitrite will react with reagents A + B and the medium will turn red. This color change will occur very quickly and can be recorded as a positive result.

    2. If the medium remains colorless there are two possibilities: 1) the nitrate was further reduced to nitrogen gas or ammonia or 2) the nitrate was not reduced at all.To differentiate between these two possibilities, zinc is added. Zinc will react with nitrate, the original substrate, and reduce it nitrite. This newly produced nitrite will then react with the reagents A + B that were added previously and turn red. If your medium turns red after adding Zinc, then the reaction is negative because the original substrate, nitrate, was not reduced. This reaction may take 5 to 10 minutes to develop.If there is no color change after adding Zinc, then the original substrate nitrate was reduced to a product that cannot be detected by reagents A + B. These products can be either N2 gas or NH3, ammonia. This is a positive result.

Therefore:

If A + B  red, then organism is positive for nitrate reduction

If A + B  no change, then add zinc

If A + B + Zinc  red, then organism is negative for nitrate reduction

If A + B + Zinc  no change, then organism is positive for nitrate reduction
Ex. 5-8: Citrate Utilization Test - This is a test to determine whether a bacterium can use citric acid as its sole source of organic carbon. Organisms that can use citrate as a carbon source produce the enzymes citrate permease and citrase. In a multi-step process, the citrate is broken down into pyruvate and CO2. The CO2is converted into sodium carbonate (NaCO3), which is alkaline. The pH indicator, bromthymol blue, changes from green to blue.

Organisms: E. coli and E. aerogenes - broth cultures

Media: Two tubes of Simmon’s citrate agar

Procedure: Inoculate the slant of the citrate tubes with the appropriate organism using the stab-streak inoculation method. In this procedure the inoculated needle is first stabbed into the bottom (butt) of the tube and then it is dragged across the slant surface of the tube. Incubate at 35° C for 48 hours or more. Make sure that the top is quite loose when these tubes are incubated. Look for a color change in the medium.
Ex. 5-13: Urease Test - This test is done to determine whether an organism has the enzyme, urease, the enzyme that breaks down urea to carbon dioxide, ammonia and water. The presence of the enzyme is indicated by a color change in the medium. The product ammonia is alkaline and causes the pH indicator, phenol red, to change color from pale orange to iridescent pink, indicating the presence of urease.

Organisms: P. vulgaris and E. coli

Media: Two tubes of urea agar

Procedure: Inoculate the agar tubes using the stab-streak method and incubate at 37° C. Observe any color change that occurs in the medium. The production of a bright pink color indicates that the organism is positive for urease production.
Ex. 5-20: SIM Test - This test is used to determine whether an organism produces hydrogen sulfide (S), whether it can metabolize the amino acid tryptophane to indole (I), pyruvic acid and ammonia, and whether or not the organism is motile (M). These three tests are all done in a single tube, a deep known as a SIM tube.

Organisms: Escherichia coli, Enterobacter aerogenes, Proteus vulgaris & Staphylococcus aureus

Media: Four SIM tubes

Procedure:

1. Using a needle, inoculate the tubes using a stab procedure. Incubate at 37° C. When you read the results, make sure you do the observations for hydrogen sulfide production (blackening of the medium) and motility (cloudiness away from the stab line) before you test for indole production.



2. Testing for indole must be done under the hood. Never use the Kovac’s reagent out in the laboratory. To your SIM tube, add 10 to 20 drops of Kovac’s reagent. You do not need to agitate or shake the tube. The Kovac’s reagents will react immediately if indole is present and produce a thin band of magenta color above the agar surface. The absence of a color change (remains yellow) indicates that indole was not produced and can be recorded as a negative result.

You must use caution with Kovac’s reagent. It is only to be used in the hood. Do not inhale the reagent or get it on your skin. Hands should be washed immediately if you should get some on your skin. Dispose of the tubes in the small red autoclave bags when you have completed this test.





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