Tutorial: Gram Negative Bacteria

PART A

1. If a Gram negative rod is isolated from a food sample on NA and there is very little history,
how might you proceed to identify this isolate to genus level?

2. Which attributes that were very useful for Gram POSITIVE bacteria from food, are not as
useful for the Gram negative bacteria?

3. What are the 2 key characteristics needed to identify Gram negative bacteria?

4. Within the genus Pseudomonas which sub-group is of greatest significance to the food
industry and why?

5. How could these species (from Q.4) be isolated from food, purified and then identified to
species level?
6. For this sub group of pseudomonads, what is more important to the food industry than their
identification to species level?

7. The Gram negative rods that are O & F positive, oxidase positive need to be further sub-
divided to genus level. Although in theory Thornley’s arginine serves this purpose, it may be
difficult to interpret this test. How should this test be set up and interpreted?

8. What additional tests should be inoculated in parallel to verify the results from Thornley’s
arginine test? How are these tests set up and interpreted?

9. If Vibrios are suspected to be in a food or water sample what selective and differential
medium would you use for enumeration and isolation? How could V.cholerae and
V.parahaemolyticus be differentiated on this medium? What additional tests would be
recommended to confirm the identification, which ones and what results would be expected?
 10. Any Gram negative rods, O & F +ve, oxidase –ve, should be set up on a selective/
differential medium before further identification is attempted. Which medium would you
recommend? How does it work and what genera can be separated based on this medium?

PART B

1. For what purposes are King’s A and B agars used in bacterial identification? How do they
differ and why do they produce the responses they do?

2. Which genera belong to the coliform group of bacteria? What can the presence of
significant numbers of coliforms in food indicate?
 3. Identify the following Gram negative bacteria based on the results presented in the table
below. Note not all tests are inoculated for all organisms.

Tests Results for organisms

A B C D E F G
Oxidase - - + - + - +
Oxidation & O&F O&F O&F O&F O only O&F O&F
Fermentation
Thornley’s arginine + + -
Moller’s ornithine - +
Moller’s lysine - +
Moller’s arginine + + -
Phenylalanine - - - - - - -
deaminase
MR - + + + - + +
VP + - + - -
Citrate + + - + - +
Gelatin hydrolysis v - - - -
Acid from sucrose + - + - -
Gas from glucose + + + + - - +
Indole - - + v
motility + + + + + - +

If these unknowns cannot be fully identified, what additional tests are needed?

Unknown A …………………………………………………………………………………

Unknown B …………………………………………………………………………………

Unknown C …………………………………………………………………………………

Unknown D …………………………………………………………………………………

Unknown E …………………………………………………………………………………

Unknown F …………………………………………………………………………………

Unknown G …………………………………………………………………………………
 4. What are the advantages of using miniaturized biochemical test strips (microbact) for
identification of bacteria?

Question 5
Who am I? Read the descriptions provided and once you have identified the microorganism, write
the genus and species name in the appropriate place. The same microorganism may be used on
more than one occasion.

a) I produce pink colonies with black centres when I grow on XLD agar.

…………………………………………………………………………………………..

b) I am an oxidase positive, oxidative and fermentative Gram negative coccoidal or rod shaped
organism that grows best in about 3% salt.

…………………………………………………………………………………………..

c) I am an oxidase negative, oxidative and fermentative Gram negative rod shaped organism that
ferments lactose in the presence of bile salts and some of my serotypes cause food poisoning but
mostly I am harmless.

…………………………………………………………………………………………..