culture methods by Dr.vimal prakash

1. CULTURE METHODS

2. PURPOSE
•Pure culture
•Biochemical tests
•Antimicrobial susceptibility testing
•Stock culture
•Sufficient growth-antigens
•Typing
•Viable bacterial counts

3. METHODS
Loops and Straight wires:
•Bacteriogical loops-2to4mm-Streaking
•Bacteriological Straight wires-Stroke and Stab
culture
•Bunsen flame-redhot-cool-10secs
•Biological safety cabinet

8. STREAK CULTURE
•Isolation
•Individual isolated –mixed culture
•Method of streaking:
•Incubation 37deg C -12to18hrs
•Biochemical tests

11. LAWN OR CARPET CULTURE
•Swabbing
•Flooding
USES:
•AST
•Bacteriophage typing
•Bacterial grth-Preparation of antigens and vaccines

13. STROKE CULTURE
Agar slopes or slants-zigzag fashion
•Diagnostic tests
•Citrate utilization test and urease test

15. STAB CULTURE
Stabbing semisolid agar butt-straight wire
•Stock culture
•OF test
•Motility testing
•Eg. MMM , Nutrient agar semisolid butts , TSI.

17. LIQUID CULTURE
Touching with a loop or
Adding the with pipettes or Syringes
Bacterial growth:
•Uniform turbidity
•Granular turbidity
•Surface pellicles
Uses:
•Blood culture
•Sterility testing
•Water analysis

19. POUR PLATE CULTURE
Viable bacterial count
•Number of bacteria per ml of liquid broth/specimen
▪Serial 10 fold dilutions
▪Pour plating
▪Colony counting
▪Viable count per ml

22. SPREAD-PLATE METHOD
Known volume of individual dilutions are
spread evenly on the surface of agar plate to
obtain lawn culture
After incubation colonies are counted
&multiplied by DF to estimate bacterial count

23. INCUBATION OF CULTURE
MEDIA
•Pathogenic organisms-37 C
•Aerobic bacteria-culture plates – 37 C
•Overnight in an incubator
•Bacteriological incubator

25. INCUBATORY CONDITIONS
•Candle jar
Capnophilic bacteria-Brucella abortus,
Streptococcus, Pneumococcus,Gonococcus
•Microaerophilic bacteria-Campylobacter,
Helicobacter
Require 5% oxygen -growth

27. ANAEROBIC CULTURE METHODS
Obligate anaerobes
•Production of vacuum-vacuum desicator
•By displacement and combustion of oxygen
MCINTOSH AND FILDES ANAEROBIC JAR: Evacuation of air from jar and
replacement with inert gas like hydrogen followed by removal of residual oxygen
by catalyst.
Aluminium pellets coated with palladium
Removal of residual oxygen
(O2+H2-------H2O)
ANOXOMAT:
Evacuation of air from the jar and replaces by hydrogen gas from a cylinder.Same
catalyst.
Effective method.

30. •ABSORPTION OF OXYGEN BY CHEMICAL METHODS
Chemical reactions
GASPAK SYSTEM
Sachet-sodium bicarbonate andsodium borohydride
Chemical indicator-reduced methylene blue
Biological indicator –Pseudomonas
•ANAEROBIC GLOVE BOX(ANAEROBIC CHAMBER)
Without O2
•BY USING VARIOUS REDUCING AGENTS
Glucose,thioglycollate,cooked meat pieces,
Robertson cooked meat broth-chopped meat
•PREREDUCED ANAEROBICALLY STERILIZED(PRAS)
Sealed foiled pockets

33. PRESERVATION OF
MICROORGANISMS
Epidemiological investigation,future research and
educational purposes.
•Short term-weeks to months
•Long term-years

34. SHORT TERM METHODS
Phenotypic and genotypic-altered
•Subculture-cookedmeat medium-anaerobes
•Mineral oil,glycerol,sterile distilled water
•Freezing at -20 deg C
•Drying-moulds,spore bearing bacteria

35. LONG TERM PRESERVATION
METHODS
Advantages
Less space,phenotypic and genotypic maintained,reduced
chance of mutation,viability well maintained.
•Ultra temperature freezing-cryopreservative agents-
glycerol,skimmed milk,sucrose and incubation at -70deg C
•Lyophilization-freezing of liquid culture followed by
dehydration to remove water from frozen bacterial suspensions
Stable readily rehydrated product-4deg C

36. METHODS OF ISOLATING
BACTERIA IN PURE CULTURE
▪Surface plating-Intermittentheat
▪Selective media and enrichment broth-eg faeces
▪Pretreatment of specimens-4%
▪Anaerobiasis
▪Heating-thermophilic 60 deg c
▪Filters-different pore diam-sizes
▪Based on motility-SC in craigie
▪Animal inoculation-guinea pigs

37. THANK YOU