Haematology and Microbiology Laboratory Guide

Question 1

Sequence of gram-staining

Answer 1

1. Immerse in Coplin Jar containing crystal violet solution. 1 minute. Rinse. Drain.
2. Immerse in Coplin Jar containing Gram’s iodine solution. 1 minute. Rinse. Drain.
3. Add drops of acetone. 3-5 seconds. (CRUCIAL)
4. Wash with water. Drain.
5. Immerse in Coplin Jar containing safranin (counter stain)

Question 2

Colour of gram +ve and -ve

Answer 2

Blue violet –> crystal violet-iodine complex (+ve)

Red –> counter stain safranin (-ve)

Question 3

Streak plate methods

Answer 3

1. Primary inoculum. Streak from solid media/broth/swab
2. Flame-sterilize
3. Streak across the original inoculum
4. Sterilize
5. Streak again.
6. DO NOT STERILIZE.
7. Last streak

*half plates: 3 streaks only

Question 4

Catalase test function

Answer 4

Distinguishing staphylococci +ve from streptococci -ve

2 types:
Direct plate (do not use on blood agar as blood contains catalase) and slide test

Question 5

Coagulase test function

Answer 5

Distinguish S.aureus which possesses coagulase (clumping factor) from other staph.

Question 6

Oxidase test function

Answer 6

Determine the presence or absence of intracellular cytochrome c oxidase enzyme. Identify gram -ve rods ie pseudomonas and vibrio (oxidase-positive : aerobic). Enterobacteriaceae (oxidase negative : anaerobic)

How it works?
Cytochrome oxidase oxidizes cytochrome c which in turn oxidizes the p-phenylalanine reagent to form a purple coloured compound.

Question 7

Lawn plate streaks

Answer 7

1. Streak the first two times without recharging the swab

2. Third streak after recharge

Question 8

Bacitracin sensitivity test function

Answer 8

Distinguish b-haemolytic streptococci group A and other b-haemolytic streptococci.

If group A, zone of inhibition >10m. If 20-30 mm indicates S.pyogenes

Question 9

Novobiocin sensitivity test

Answer 9

A rapid test to distinguish Staphylococcus saprophyticus from other coagulase-negative staphylococci. (S) Zone of inhibition ~20/30mm –> CONS. (R) Growth up to edge of disc –> S. saprophyticus

Question 10

Optochin sensitivity test

Answer 10

Distinguish Strep pneumoniae (S) from alpha-haemolytic streptococci.