Diagnostic Bacteriology

Question 1

How does gram-staining work?

Answer 1

Identifies 2 groups of bacteria:
Gram negative and gram positive

1. Application of crystal violet
2. Application of iodine- formation of crystal violet and iodine complex
3. Alcohol wash- alcohol dehydrates peptidoglycan layer
4. Application of safranin- stains pink

Question 2

What is gram negative bacteria?

Answer 2

Has 2 outer membranes with thin peptidoglycan

Stains pink

Question 3

What is gram positive bacteria?

Answer 3

has 1 outer membrane with thick peptidoglycan

Stains purple

Question 4

What shapes can bacteria be?

Answer 4

Spheres= cocci
Diplococci- 2 cocci
Diplococci encapsulates- 2 cocci encapsulated
Staphylococci- many cocci in group
Streptococci- many cocci in line
Sarcina- 3D cube cocci formation
Tetrad- square cocci formation

Cylinders= bacilli
Coccobacillus- oval shape
Diplobacilli- 2 bacilli
palisades- bacilli joined by long side
streptobacilli- bacilli in a line (chain)

Question 5

What does a hemolytic activity growth test show?

Answer 5

Gamma haemolysis= no haemolysis (no zone)
Alpha Haemolysis= partial haemolysis (opaque zone)
Beta haemolysis= complete haemolysis (transparent zone)

Question 6

How do you carry out a hemolytic activity growth test?

Answer 6

1. single colony picked up from agar plate
2. streak out bacteria colony on agar plate with 5% sheep RBCs
3. Incubate agar overnate and assess haemolytic activity the next day

Question 7

What does a lactose fermentation growth test involve?

Answer 7

Growth on MacConkey agar containing bile sales, crystal violet and lactose
tests capacity of a microorganism to ferment lactose

Question 8

What does a lactose fermentation growth test show?

Answer 8

No growth- bile salts inhibit growth of gram-positive bacteria
Lactose fermenting- bacteria produce acid during lactose fermentation
Lactose non-fermenting- bacteria don’t ferment lactose
Difference in acidity is measured to determine if its lactose fermenting

Question 9

How is a lactose fermentation growth test carried out?

Answer 9

1. Pick bacteria colony if agar plate
2. Streak out bacteria onto MacConkey agar plate that contains pH indicator (agar is red at neutral)
3. Incubate plate overnight and assess in morning

Question 10

What is a catalase test?

Answer 10

Determines if a bacteria produces catalase- an enzyme that catalyses the breakdown of hydrogen peroxide into O2 and H2O

Question 11

What does a catalase test show us?

Answer 11

Catalase + = O2 bubbles

Catalase - = no O2 bubbles

Question 12

How is a catalase test carried out?

Answer 12

1. bacteria is applied to a glass slide
2. Hydrogen peroxide is applied to bacteria
3. Short incubation period then observe for bubbles

Question 13

What is a coagulase test?

Answer 13

Determines whether a bacteria produces coagulase- an enzyme that cross links fibrinogen to form a clot on bacterial surface
It can determine S. aures (produces coagulase) from other staphylococci ( dont produce coagulase)

Question 14

What does a coagulase test show us?

Answer 14

Coagulase + = clumps

Coagulase - = no clumps

Question 15

How is a coagulase test carried out?

Answer 15

1. Bacteria applied to slide
2. Plasma is applied on top
3. This is shortly incubated with gentle rotation
   4 Look for clumps