Diagnostic Bacteriology

Question 1

Why is accurate diagnosis of pathogens necessary?

Answer 1

To identify appropriate treatments

To prevent and manage outbreaks

Prevent over use or Mis use of treatments

Question 2

What are the steps a doctor makes in making a diagnosis?

Answer 2

Diagnosis at the clinic - using patient history and physical

Nom-microbial investigations - radiology and haematology

Laboratory investigations - lots of tests

Question 3

What is the principal approach of microscopy in terms of bacteria?

Answer 3

The gram stain

Question 4

What is the gram stain?

Answer 4

Identifies two groups of bacteria based on the physical and chemical compositions of their cell wall

GRAM NEGATIVE - 2 outer membranes with thin peptidoglycan between them (stains pink)

GRAM POSITIVE - 1 outer membrane with thick peptidoglycan outside the membrane (stains purple)

Question 5

What colour does gram negative bacteria stain?

Answer 5

Pink

Question 6

What colour does gram positive bacteria stain?

Answer 6

Purple

Question 7

How is a gram stain prepared and analysed?

Answer 7

1. Bacteria is attached to the glass slide. The culture is spread on the slide and fixed with heat
2. Crystal violet (purple) is added on top of the bacteria. These molecules can enter the bacterial cell wall
3. Iodine solution is added. Forms crystal violet-iodine complexes, these are larger than the origkn ones
4. Alcohol wash. Dehydrates the peptidoglycan layer, tightening it. In gram positive bacteria, as the layer is thick and now tight, the complexes can’t be washed out. So remain purple. In Gram negative bacterial, as the layer is thin, the complexes are washed out, so these decolourise
5. Safranin (pink) counterstain. Stains gram negative bacteria pink. Positive stay purple

Question 8

Apart from type what other information can the gram stain provide?

Answer 8

Morphology

Question 9

What do the shapes of bacteria mean (on a gram stain)

Answer 9

Spheres = coccus

Round ended cylinders = bacillus

Staphylococci are like a bunch of grapes
Streptococci are chains of spheres

Question 10

Looking at the haemolytic activity of a bacteria is a culture based method. What does it differentiate between?

Answer 10

Gram positive species such as staphylococci, streptococci and enterococci

Question 11

What does the test of haemolytic activity test?

Answer 11

The capacity to produce haemolysins

Gamma-haemolysis = no haemolysis

Alpha haemolysis = partial

Beta haemolysis = complete haemolysis

Question 12

How does the test for haemolytic activity work?

Answer 12

1. A bacterial colony is chosen
2. It is spread onto an agar plate that has a medium that contains 5% sheep’s red blood cells
3. The bacteria are incubated overnight
4. Gamma = no zone
   Alpha = opaque zone
   Beta = transparent zone

Question 13

What are the four culturing techniques?

Answer 13

Haemolytic activity

Lactose fermentation

Catylase test

Coagulase test

Question 14

What is lactose fermentation used to distinguish between?

Answer 14

Gram negative

Question 15

How does the lactose fermenting test work

Answer 15

Growth on macConkey agar containing bile salts, crystal violet and lactose

No growth = bile salts inhibit production of gram positives

Lactose fermenting = have lactase so produce lactic acidosis during fermentation (pink)

Lactose non fermenting = do not ferment lactose (yellow/colourless)

The difference in acidity is measured

Question 16

How is the lactose fermentation test performed?

Answer 16

1. Pick a bacterial colony
2. Streak bacteria onto MacConkey agar that contains a pH indicator
   (Neutral =red)
3. Incubate overnight
4. Lactose fermenters, lowers ph, goes pink.
   Non fermented, colourless colonies, yellow

Question 17

What does the catalase test test for?

Answer 17

Distinguishes staphylococci and streptococci based on the production of catalase, an enzyme that breaks down hydrogen peroxide into oxygen and water

Staphylococci = catalase (bubbles)

Streptococci = no catalase (no bubbles)

Question 18

How is the catalase test performed?

Answer 18

1. Apply bacteria onto glass slide
2. Apply hydrogen peroxide
3. Short incubation
4. Bubbles or no bubbles?

Question 19

What does the coagulase test distinguish?

Answer 19

Differentiates staphylococcus aureus (clumps) from others staphylococci

Determines whether a bacteria produces coagulase, an enzyme that processes prothrombin into thrombin, cleaving fibrinogen into fibrin. Factor XIIIa then forms cross links in fibrin in plasma to form a clot

Coagulase = clumps
No coagulase = no clumps

Question 20

How is the coagulase test performed?

Answer 20

1. Bacteria applied to a glass slide
2. Plasma applied on top
3. Short incubation
4. Generation of clumps?