General bacteriology Flashcards
Purposes of laboratory diagnosis of bacterial infection
- Identification
- treatment
- surveillance purpose
- for outbreak investigation
- to start PEP (Post exposure prophylaxis)
- to initiate appropriate infection control measures
The different types of laboratory diagnosis of bacterial infections
- Specimen collection
- Direct detection (microscope, antigen detection, molecular diagnosis)
- culture
- identification (bacterial identification, automated identification)
- antimicrobial susceptibility testing
- serology
- molecular methods
- typing methods
Louis Pasteur and Robert Koch in culture media development
Louis Pasteur developed liquid media/ broth
Rapid growth
Robert Koch introduced solid media using gelatin (15%)
Visible colonies
Disadvantages of gelatin as solidifying medium
- Proteolysed by some bacteria
- Solid only at temperatures less than 24°C
15% gelatin was used
Other solidifying agents are serum and egg
Agar agar properties
Extracted from sea weeds and red algae Also called Chinese grass Polysaccharide containing: 70% agarose 30% agaropectin Inorganic phosphate and calcium
Agar agar and its concentration
- 2 - 2 % for solid media (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
Methods to inhibit swarming of bacteria
- Firm agar (5-6%)
- CLED (cysteine lactose electrolyte deficient) or MacConkey agar
- Add alcohol, boric acid, bile salts, chloral hydrate, sulfonamides, sodium azide, surface acting agents
Gram positive bacteria that shows swarming
Clostridium tetani Bacillus cereus (‘serious’)
Gram negative bacteria that shows swarming
Proteus vulgaris
Proteus mirabilis
Vibrio alginolyticus
Vibrio parahemolyticus
Simple or basal culture media
Contains only sources of C and N for the nutritionally non-fastidious bacteria
- Peptone water
- Meat extract (+peptone water)
- Agar (+ meat extract +. ) or nutrient agar
Enriched culture media
- 5% sheep blood agar
- Chocolate agar
Egg mediums (for mycobacterium) like: - LJ medium
- Dorset egg medium
Serum containing medium like - Loeffler’s serum slope for C. diphtheriae
- PPLO broth/agar
Blood agar production
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)
Selective culture media
- Change pH
- Adding antibiotics
- Chemical agents
- Dye
pH of culture media and bacteria
For pathogenic bacteria it is usually 7.2-7.4
For vibrio it is 8.2-8.4
Example TCBS medium
Culture media for Neisseria
Thayer Martin medium contains: vancomycin Colistin Nystatin while modified Thayer Martin medium contains trimethoprim in addition to the other antibiotics
PPLO medium contains
Penicillin
MacConkey medium
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
DCA medium
Contains sodium deoxycholate citrate for salmonella and shigella
Potassium tellurite and culture medium
All corynebacterium are resistant to 0.3-0.4% concentration of Potassium tellurite
Example tinsdale medium
All staphylococcus are resistant to
7-10% salt
Example: salt milk agar
Potassium tellurite and culture medium
All corynebacterium are resistant to 0.3-0.4% concentration of Potassium tellurite
Example tinsdale medium
Eosin methylene blue agar
Selective media for gram negative bacteria
LJ culture medium
Selective media for mycobacterium
Malachite green dye
Examples of enrichment media
- Alkaline peptone water for vibrio
2. Selenite F broth for salmonella and shigella
Differential medium
Based in colony morphology and colour
Examples:
1. Blood agar
2. MacConkey medium
Blood agar
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
TCBS medium
Differentiates sucrose fermenting vibrio from non sucrose fermenting vibrio
Bromothymol blue gives yellow colour to sucrose fermenters
Mannitol salt agar
Mannitol fermenting staphylococcus (yellow) and non mannitol fermenting staphylococcus (pale) are differentiated
CLED medium
Cysteine lactose electrolyte deficient medium
Lactose fermenting urinary pathogens (yellow) from non lactose fermenting primary pathogens (pale)
Hugh Leifson oxidative fermenting medium
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
Indicator media examples
- MacConkey media: neutral red
- TCBS medium: bromothymol blue
- Hugh Leifson medium: bromothymol blue
Transport media
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
Standard inoculum of test bacterium
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
Methods of sensitivity testing classification
1. Dilution (reference method: Agar dilution Broth dilution Microbroth dilution Macrobroth dilution 2. Disc diffusion 3. E test
Broth dilution method
Sensitivity testing
Nutrient broth medium used
Standard inoculum:
Overnight broth culture in peptone water
Interpretation of dilution method
Resistant:
MIC> breakpoint value
Sensitive:
MIC value < breakpoint value
Disadvantages of broth dilution method
- Cumbersome and laborious
2. Fastidious organisms cannot be tested
Agar dilution method
Media, advantages and disadvantages
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
Kirby Bauer disc diffusion method
Mueller Hinton agar
6 discs impregnated with standardised concentration of antibiotics
Very easy to do
Cannot exactly determine MIC
Stoke’s method
Control strain and test strain are incubated in the same petridish
Easy to do
Does not determine MIC
Epsilometer test out E test
Combination of dilution and disc diffusion method
Easy and quantitative test
Determine MIC
