Diagnostic bacteriology

Question 1

What are the differences in gram + and - bacteria - membranes?

Answer 1

• : 2 outer membranes with thin peptidoglycan

+ : 1 outer membrane with thick peptidoglycan

Question 2

How to differentiate + and - with stain?

Answer 2

Apply crystal violet

Apply Iodine

Alcohol wash

Apply safranin

• negative looks pink -doesn’t retain crystal violet only safranin
• positive looks purple

Question 3

What are the basic morphology of bacteria?

Answer 3

Sphere - coccus

Round ended cynlinders - Bacilli

Question 4

How to differentiate straphylococcus, streptococcus, enterococcus?

Answer 4

Using their hemolytic activity:

Gamma hemolysis : no hemolysis, no zone
Alpha hemolysis : partial , opaque zone
beta hemolysis : complete hemolysis, transparent zone

Question 5

How to use lactate fermentation to differentiate negative bacteria? ( bacilli )

Answer 5

Grow on McConkey agar- has bile salts and crystal violet

• lactose fermenting bacteria : produce acid
• lactose non-fermenting : do not ferment lactose

Colour change on agar- fermenter will turn pink
non fermenter - colourless

Question 6

Why cannot you use lactate fermentation for gram positive bacteria?

Answer 6

Bile salts inhibits growth of positives

Question 7

How to differentiate between gram positive bacteria?

Answer 7

Catalase test:

Whether a bacteria produces catalase
Catalase : can see oxygen bubbles

Apply bacteria to glass slide
Apply hydrogen peroxide
Observe the slide

Question 8

What catalase results differentiate staphylococci and streptococci?

Answer 8

staphylococci - produces catalase

streptococci - does not produce catalase

Question 9

What test can further differentiate staphylococci?

Answer 9

Coagulase test

Coagulase is an enzyme that cross-links fibrinogen to form a clot:

S.Aureus = positive test - clumps
Other staphylococcus = negative test

Apply bacteria to slide
Apply plasma
Incubate 15 secs
Observe slide