Microbiology

Question 1

What colour is Gram positive and negative bacteria?

Answer 1

Gram positive - deep violet

Gram negative - pink

Question 2

How to prepare a gram stain

Answer 2

1. Pick a bacterial
   colony from one agar plate using a sterile plastic loop
   and smear some bacteria onto the slide.
2. Dry gently with a hair dryer.
3. Add a few drops of crystal violet
4. Gently rinse slide with tap water
5. Cover the slide with lugol’s iodine
6. Gently rinse slide with tap water.
7. Cover the slide with alcohol
8. Immediately rinse slide with tap water.
9. Cover the slide with a few drops of safranin
10. Gently rinse slide with tap water.
11. Blot any excess liquid with tissue and then inspect under the microscope.

Question 3

What should be observed under the microscope?

Answer 3

• colour
• shape
• clustering, chains or single?

Question 4

How can blood agar plates be useful?

Answer 4

Some pathogens produce haemolysins, substances that lyse/destroy red blood cells in blood-containing media, in a process called haemolysis. They can produce different types of haemolysis also.

Question 5

How can MacConkey agar plates be used?

Answer 5

• If the plate is red/pink then the bacteria is a lactose
  fermenter.
• If the plate is colourless/yellow then the bacteria are lactose non-fermenter

Question 6

What is the oxidase test?

Answer 6

The oxidase test is used to determine if an organism produces the enzyme cytochrome c oxidase. Oxidase positive bacteria are aerobic and can use oxygen as an electron acceptor during respiration.

Question 7

How does the oxidase test work?

Answer 7

The test strip contains TMPD which is a redox indicator and acts as an artificial electron acceptor in this test. It changes from dark blue/purple when oxidized and colourless when reduced.
Strips of paper used in the test and rubbed bacterial colonies and colour is observed.

Question 8

What is the catalase test?

Answer 8

Hydrogen peroxide is a toxic end-product of the aerobic breakdown of sugars. Catalase is a common enzyme found in many living organisms and can break down hydrogen peroxide into gaseous oxygen.

Question 9

How can the catalase test be used?

Answer 9

1. You are provided with hydrogen peroxide
2. Pick a loopful of bacteria using the plastic loop and mix it in the hydrogen peroxide
3. Do you see bubbles? If so, then the bacteria are catalase positive

Question 10

What is the coagulase test?

Answer 10

This test checks for the presence of the enzyme coagulase (a clumping factor) which reacts with fibrinogen to form clumps or clots. The coagulase test is used to distinguish different types of Staphylococcus.

Question 11

How is the coagulase test performed?

Answer 11

1. Shake the test reagent from the kit thoroughly.
2. Add 1 drop of the Test reagent to the test circle.
3. Using a plastic loop, pick bacterial colonies and mix with the reagent in the test circle.
4. Do you see clumping/clots? If so, then the bacteria are coagulase positive. This indicates the presence of Staphylococcus aureus.

Question 12

How can antibiotic sensitivity be measured?

Answer 12

1. Measuring the zone of inhibition surrounding antibiotic discs on a bacterial plate
2. Using the E-Test to measure the Minimum inhibitory concentration (MIC) for a specific antibiotic

Question 13

How can antibiotic sensitivity be used?

Answer 13

Determine if a bacteria is resistant to a particular antibiotic and compare to a table

Question 14

How is the coefficient of sensitivity measured?

Answer 14

diameter of zone of inhibition - sensitive zone