Monday, August 20, 2012

Biochemical Tests for Identifying Unknowns

Carbohydrate Utilization:
Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the media remains red. If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it.
The carbohydrate tests we perform are the:
  1. Glucose (Dextrose) test
  2. Lactose test
  3. Sucrose test
All carbohydrate test media should be inoculated with the transfer loop.
Fermentation results from left to right:
  • Left tube shows less acid formation than far right tube, but gas is still made
  • Center shows no carbohydrate utilization to produce acid or gas.
  • Right tube shows acid was produced as evidenced by the yellow color, and gas was made (look at the bubble in the Durham tube)

Citrate Utilization:
Tests for the ability of bacteria to convert citrate (an intermediate of the Kreb’s cycle) into oxaloacetate (another intermediate of the Kreb’s cycle). In this media, citrate is the only carbon source available to the bacteria. If it can not use citrate then it will not grow. If it can use citrate, then the bacteria will grow and the media will turn a bright blue as a result of an increase in the pH of the media. To inoculate this slant, use the transfer loop.
 Left tube is a negative result. Right tube is a positive result.

Gelatin Utilization:
This media is used to test if bacteria can digest the protein gelatin. To digest gelatin, the bacteria must make an enzyme called gelatinase. To inoculate this media, use a transfer needle to stab the gelatin. After incubating the inoculated media for at least 48 hrs, transfer the tube into a refrigerator. The tube should be completely chilled prior to observation. If the media is solid after refrigeration then the test is negative (the bacteria did not digest gelatin). If the media is liquefied even after refrigeration, then the test result is positive…the bacteria is able to digest gelatin.

The 'Serratia marcescens' on the left is positive for gelatinase production, as evidenced by the liquidation of the media.  The 'Salmonella typhimurium' on the right is negative, as evidenced by the solidity of the media.

Starch hydrolysis:
This test is used to detect the enzyme amylase, which breaks down starch. After incubation the plate is treated with Gram’s iodine. If starch has been hydrolyzed (broken down) then there is a reddish color or a clear zone around the bacterial growth; if it has not been hydrolyzed then there is a black/blue area indicating the presence of starch. Simply use inoculating loop to spread bacteria onto plate surface. After the bacteria have grown, you add a few drops of Gram’s iodine to the plate and look for the color immediately after adding the iodine.

Indole production:
This test is done to determine if bacteria can breakdown the amino acid tryptophan into indole. SIM media or TSB (tryptic soy broth) is inoculated using a transfer needle. After incubating the bacteria for at least 48 hours, Kovac’s reagent is added to the media to detect if indole has been made by the bacteria. The development of a red/pink layer on top of the media is a positive result (the bacteria can breakdown tryptophan to form indole). Failure to see a red layer is a negative result (indole was not formed from tryptophan).

The tube on the left with the red ring is positive for indole production while the tube on the right shows a negative result.

The tube on the left is negative and the tube on the right is a positive result.

MRVP (methyl red-Vogues Proskauer):
This test is used to determine two things. The MR portion (methyl red) is used to determine if glucose can be converted to acidic products like lactate, acetate, and formate. The VP portion is used to determine if glucose can be converted to acetoin.
These tests are performed by inoculating a single tube of MRVP media with a transfer loop and then allowing the culture to grow for 3-5 days. After the culture is grown, about half of the culture is transferred to a clean tube. One tube of culture will be used to conduct the MR test, the second tube serves as the VP test.
  1. MR (methyl red) test:
  2. Methyl red is added to the MR tube. A red color indicates a positive result (glucose can be converted into acidic end products such as lactate, acetate, and formate. A yellow color indicates a negative result, glucose is converted into neutral end products.
  3. VP (Vogues Proskauer) test: First alpha-napthol (also called Barritt’s reagent A) and then potassium hydroxide (also called Barritt’s reagent B) are added to the VP tube. The culture should be allowed to sit for about 15 minutes for color development to occur. If acetoin was produced then the culture turns a red color (positive result); if acetoin was not produced then the culture appears yellowish to copper in color (a negative result).

Triple sugar Iron (TSI) - & Hydrogen sulfide production (H2S): Looks at fermentation of glucose, lactose, and sucrose and checks if hydrogen sulfide is produced in the process. Basically a pH indicator will change the color of the media in response to fermentation…where that color change occurs in the tube will indicate what sugar or sugars were fermented. The presence of a black color indicates that H2S was produced. In this media, H2S reacts with the ferrous sulfate in the media to make ferrous sulfide…which is black. To inoculate, use a needle to stab agar and then uses a loop to streak the top slated region.
In addition to TSI media, SIM media can be used to determine if H2S is produced. A black color in the SIM medium following inoculation and incubation indicates that H2S is made by the bacteria.

does not ferment either lactose or sucrose
ferments lactose and/or sucrose

REDno fermentation of glucose
YELLOWsome fermentation of glucose has occurred, acid has been produced
GAS FORMEDSeen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube.  (Caution:these gassy fermenters may have bacteria close to the opening.)
BLACKH2S has been produced

From left to right:
  1. Uninoculated control
  2. Red slant and red butt, no black color= no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced
  3. Red slant and black butt= no lactose or sucrose fermentation, H2S has been produced
  4. Red slant with yellow butt= no lactose or sucrose fermentation, lactose is fermented, no H2S has been produced
  5. Yellow slant, yellow butt and black coloration= Lactose, sucrose and glucose fermented, and H2S has been produced
  6. Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced
  7. Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas been produced

This test is used to detect the enzyme urease, which breaks down urea into ammonia. Ammonia is a base and thus will raise the pH of the media if it is present. This change in pH is indicated by a pH indicator called phenol red which is present in the media. A color change from yellow to bright pinkish-red is positive; lack of color change is a negative result. Inoculate the liquid media with a transfer loop.

The tube on the left is a positive reaction; the tube in the middle is a negative reaction and the tube on the right in an un-inoculated control.

This test is can be used to detect the enzyme catalase. This enzyme is responsible for protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the organism. Catalase breaks H2O2 down into water and O2. To perform the catalase test simply smear a small amount of the test organism onto the lid of a Petri plate/culture dish. Then add a drop of hydrogen peroxide to the smear. If bubbles become visible (these would be the O2 bubbling up) then the test is positive and you can conclude that the organism makes catalase. A lack of bubbles indicates the absence of catalase. *Note, most aerobic organism make catalase.

Bubbling upon the addition of hydrogen peroxide is indicative of the presence of catalase for this organism.

Oxidase test:
To perform this test simply swab some of your test culture into one of the boxes on an oxidase dry slide. If a color change to purple or blue is evident at 30 seconds-1 minute then the result is positive. It is important that the test is read by one minute to ensure accurate results (avoid false negatives and false positives). This laboratory test is based on detecting the production of the enzyme cytochrome oxidase by Gram-negative bacteria. It is a hallmark test for the Neiserria. It is also used to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and other Enterobacteriaciae.

Additional Non-Biochemical Tests:

Motility test:
The motility test is not a biochemical test since we are not looking at metabolic properties of the bacteria. Rather, this test can be used to check for the ability of bacteria to migrate away from a line of inoculation thanks to physical features like flagella. To perform this test, the bacterial sample is inoculated into SIM or motility media using a needle. Simply stab the media in as straight a line as possible and withdraw the needle very carefully to avoid destroying the straight line. After incubating the sample for 24-48 hours observations can be made. Check to see if the bacteria have migrated away from the original line of inoculation. If migration away from the line of inoculation is evident then you can conclude that the test organism is motile (positive test). Lack of migration away from the line of inoculation indicates a lack of motility (negative test result).

Left tube is result for a non-motile bacterium. Right tube is the result for a motile organism.

Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123, ehab fathy aboueladab
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