General bacteriology

Question 1

Purposes of laboratory diagnosis of bacterial infection

Answer 1

1. Identification
2. treatment
3. surveillance purpose
4. for outbreak investigation
5. to start PEP (Post exposure prophylaxis)
6. to initiate appropriate infection control measures

Question 2

The different types of laboratory diagnosis of bacterial infections

Answer 2

1. Specimen collection
2. Direct detection (microscope, antigen detection, molecular diagnosis)
3. culture
4. identification (bacterial identification, automated identification)
5. antimicrobial susceptibility testing
6. serology
7. molecular methods
8. typing methods

Question 3

Louis Pasteur and Robert Koch in culture media development

Answer 3

Louis Pasteur developed liquid media/ broth
Rapid growth
Robert Koch introduced solid media using gelatin (15%)
Visible colonies

Question 4

Disadvantages of gelatin as solidifying medium

Answer 4

1. Proteolysed by some bacteria
2. Solid only at temperatures less than 24°C
   15% gelatin was used

Other solidifying agents are serum and egg

Question 5

Agar agar properties

Answer 5

Extracted from sea weeds and red algae
Also called Chinese grass
Polysaccharide containing: 
70% agarose 
30% agaropectin
Inorganic phosphate and calcium

Question 6

Agar agar and its concentration

Answer 6

1. 2 - 2 % for solid media (2%)
2. 2-0.5 % soft agar medium for checking motility of bacteria, Craigie’s tube (u-tube)

5-6% firm agar medium for inhibition for swarming

Question 7

Methods to inhibit swarming of bacteria

Answer 7

1. Firm agar (5-6%)
2. CLED (cysteine lactose electrolyte deficient) or MacConkey agar
3. Add alcohol, boric acid, bile salts, chloral hydrate, sulfonamides, sodium azide, surface acting agents

Question 8

Gram positive bacteria that shows swarming

Answer 8

Clostridium tetani
Bacillus cereus (‘serious’)

Question 9

Gram negative bacteria that shows swarming

Answer 9

Proteus vulgaris
Proteus mirabilis
Vibrio alginolyticus
Vibrio parahemolyticus

Question 10

Simple or basal culture media

Answer 10

Contains only sources of C and N for the nutritionally non-fastidious bacteria

1. Peptone water
2. Meat extract (+peptone water)
3. Agar (+ meat extract +. ) or nutrient agar

Question 11

Enriched culture media

Answer 11

1. 5% sheep blood agar
2. Chocolate agar
   Egg mediums (for mycobacterium) like:
3. LJ medium
4. Dorset egg medium
   Serum containing medium like
5. Loeffler’s serum slope for C. diphtheriae
6. PPLO broth/agar

Question 12

Blood agar production

Answer 12

5 mL sterile sheep blood and 95 mL of autoclave nutrient agar is cooled to 50-55•C (70-75•C in case of chocolate agar)

Question 13

Selective culture media

Answer 13

1. Change pH
2. Adding antibiotics
3. Chemical agents
4. Dye

Question 14

pH of culture media and bacteria

Answer 14

For pathogenic bacteria it is usually 7.2-7.4
For vibrio it is 8.2-8.4
Example TCBS medium

Question 15

Culture media for Neisseria

Answer 15

Thayer Martin medium contains:
vancomycin
Colistin
Nystatin 
while modified Thayer Martin medium contains trimethoprim in addition to the other antibiotics

Question 16

PPLO medium contains

Answer 16

Penicillin

Question 17

MacConkey medium

Answer 17

Contains bile salts
1. Makes it selective for gram negative bacteria
2. Differentiate between lactose fermenting (pink/magenta) and non lactose fermenting (pale) gram -ve bacteria
Indicator: neutral red

Question 18

DCA medium

Answer 18

Contains sodium deoxycholate citrate for salmonella and shigella

Question 19

Potassium tellurite and culture medium

Answer 19

All corynebacterium are resistant to 0.3-0.4% concentration of Potassium tellurite
Example tinsdale medium

Question 20

All staphylococcus are resistant to

Answer 20

7-10% salt

Example: salt milk agar

Question 21

Potassium tellurite and culture medium

Answer 21

All corynebacterium are resistant to 0.3-0.4% concentration of Potassium tellurite
Example tinsdale medium

Question 22

Eosin methylene blue agar

Answer 22

Selective media for gram negative bacteria

Question 23

LJ culture medium

Answer 23

Selective media for mycobacterium

Malachite green dye

Question 24

Examples of enrichment media

Answer 24

1. Alkaline peptone water for vibrio

2. Selenite F broth for salmonella and shigella

Question 25

Differential medium

Answer 25

Based in colony morphology and colour Examples: 1. Blood agar 2. MacConkey medium

Question 26

Blood agar

Answer 26

``` Differentiate between haemolysis types 1. Alpha haemolysis: Green or partial haemolysis 2. Beta haemolysis: Clear yellow with complete haemolysis 3. Gamma haemolysis: No haemolytic you can see the colour of colonies and the media ```

Question 27

TCBS medium

Answer 27

Differentiates sucrose fermenting vibrio from non sucrose fermenting vibrio Bromothymol blue gives yellow colour to sucrose fermenters

Question 28

Mannitol salt agar

Answer 28

Mannitol fermenting staphylococcus (yellow) and non mannitol fermenting staphylococcus (pale) are differentiated

Question 29

CLED medium

Answer 29

Cysteine lactose electrolyte deficient medium | Lactose fermenting urinary pathogens (yellow) from non lactose fermenting primary pathogens (pale)

Question 30

Hugh Leifson oxidative fermenting medium

Answer 30

Differentiates the utilisation of glucose by bacteria 1. Oxidative utilisation 2. Fermentative utilisation (both oxidative and not) 3. No utilisation pH indicator bromothymol blue and glucose are found For anaerobic condition petroleum gel is used Indicator: bromothymol blue

Question 31

Indicator media examples

Answer 31

1. MacConkey media: neutral red 2. TCBS medium: bromothymol blue 3. Hugh Leifson medium: bromothymol blue

Question 32

Transport media

Answer 32

Maintain the original count Do not allow commensals to overgrow 1. VR medium: vibrio 2. Pike’s medium: S. pyogenes in throat swabs 3. Thioglycolate broth for anaerobic bacteria 4. Cary Blair medium: universal stool transport medium

Question 33

Standard inoculum of test bacterium

Answer 33

Special suspension of test bacterium with a specified turbidity (in a units called Macfarland or McF) Temperature of incubation: 36- 37°C Time of result interpretation: 16-18 hours

Question 34

Methods of sensitivity testing classification

Answer 34

``` 1. Dilution (reference method: Agar dilution Broth dilution Microbroth dilution Macrobroth dilution 2. Disc diffusion 3. E test ```

Question 35

Broth dilution method

Answer 35

Sensitivity testing Nutrient broth medium used Standard inoculum: Overnight broth culture in peptone water

Question 36

Interpretation of dilution method

Answer 36

Resistant: MIC> breakpoint value Sensitive: MIC value < breakpoint value

Question 37

Disadvantages of broth dilution method

Answer 37

1. Cumbersome and laborious | 2. Fastidious organisms cannot be tested

Question 38

Agar dilution method | Media, advantages and disadvantages

Answer 38

Cation adjusted Mueller Hinton agar For both fastidious (add blood) and non fastidious media For anaerobic bacteria, Wilkren Chalgren agar Still cumbersome procedure but MIC can be calculated

Question 39

Kirby Bauer disc diffusion method

Answer 39

Mueller Hinton agar 6 discs impregnated with standardised concentration of antibiotics Very easy to do Cannot exactly determine MIC

Question 40

Stoke’s method

Answer 40

Control strain and test strain are incubated in the same petridish Easy to do Does not determine MIC

Question 41

Epsilometer test out E test

Answer 41

Combination of dilution and disc diffusion method Easy and quantitative test Determine MIC