CULTURE TECHNIQUES – DIAGNOSTIC TOOLS IN MEDICAL MICROBIOLOGY 

1. CULTURE TECHNIQUES –
DIAGNOSTIC TOOLS IN
MEDICAL MICROBIOLOGY
Dr. Shikha Thakur
Assistant Professor,
Department of Biotechnology,
Thakur College of Science and Commerce,
Mumbai

2. CULTURE AND THE MEDIUM
Culture : It is the term given to microorganisms that are cultivated in the lab for the purpose of
identifying and studying them.
Cultivation :
The propagation of living organisms, applied especially to the growth of microorganisms or
other cells in artificial media.
Medium :
Is the term given to the combination of ingredients that will support the growth and
cultivation of microorganisms by providing all the essential nutrients required for the growth
(i.e. multiplication) in order to cultivate these microorganisms in large number to study them.

3.  Microbiological culture medium : which are used for growing
microorganisms, such as bacteria or yeast.
 The most common growth media for microorganisms are nutrient broths
and agar plates
 Specialized media are sometimes required for microorganism and cell
culture growth.
CULTURE MEDIA
 A culture medium is a solid or liquid preparation used to grow, transport, and store
microorganisms.

4. Can be used
Enrich the numbers of bacteria.
Select for certain bacteria and suppress others.
Differentiate among different kinds of bacteria.
 Isolation, identification and long-term storage of pure cultures
BASIC REQUIREMENT OF CULTURE MEDIA
When culturing bacteria, it is very important to provide similar environmental
and nutritional conditions that exist in its natural habitat.

5. CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS)
Nutrients- water, source of Carbon and energy, source of Nitrogen
Mineral salts –Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca.
Organic growth factor like factors- ryptophan for Salmonella typhi-
X & V factors for H. influenzae
Vitamins (e.g. folic acid, vitamin B-12, vitamin K)
Oxygen
PHYSICAL REQUIREMENTS
A suitable pH
Temperature
Hydrostatic Pressure
 Osmotic pressure

6. Preparation and storage of culture media
Wash hands & wear gloves
• Sterilize all equipments
• Pour D/W in glassware (required amount)
• Add powder ingredients (required amount)
• Heat to dissolve completely
• Autoclave
• Dispense the medium into tubes ,bottles &plates
• Store at required temperature
• Sterilizing Culture Media

7. NEED FOR CULTURE MEDIA
 It is usually essential to obtain a culture by growing the organism in an artificial medium.
 If more than one species or type of organism are present each requires to be carefully
separated or isolated on culture media and obtained as pure culture for study .
 Several organism need the determination of Antibiotic sensitivity pattern for optimal
antibiotic selection
 Medium → Nutrients → support growth
TYPES OF INFECTIONS THAT CAN BE DIAGNOSED
Diagnostic cultures are commonly used to identify infectious microbes from samples
isolated from urine ( urinary tract infections), stool (diarrheal and food-borne diseases),
genital tract ( STD), throat ( strep throat), and skin (skin infections).

8. HISTORY
The original media used by Louis Pasteur – urine or meat broth
Liquid medium – diffuse growth
Solid medium – discrete colonies.
Colony – macroscopically visible collection of millions of bacteria
originating from a single bacterial cell.
Cooked cut potato by Robert Koch(1881) – earliest solid medium
Gelatin – not satisfactory
- liquefy at 24oC

9. Agar(1882)
Frau Hesse
Used for preparing solid medium
It is suphated polymercomposedof mainly D-galactose,
anhydro-L galactose and D-glucuronic acid
Obtained from seaweeds ( red algae)- Gelidium.
No nutritive value
Not affected by the growth of the bacteria.
Melts at 96oC & sets at 42oC
2% agar is employed in solid medium

10. CLASSIFICATION
BASED ON
PHYSICAL
STATE( based
on their
consistency)
BASED ON
PRESENCE OF
MOLECULAR
OXYGEN AND
REDUCING
SUBSTANCES
BASED ON
NUTRITIONAL
FACTORS
LIQUID MEDIA AEROBIC MEDIA SIMPLE MEDIA
SEMISOLID MEDIA ANAEROBIC MEDIA COMPLEX MEDIA
SOLID MEDIA SYNTHETIC MEDIA
SPECIAL MEDIA
CLASSIFICATION

11. SPECIAL MEDIA
A. ENRICHED MEDIA
B. ENRICHMENT MEDIA
C. SELECTIVE MEDIA
D. DIFFERENTIAL MEDIA
F. TRANSPORT MEDIA

12. PHYSICAL TYPE CULTURE MEDIA
Solid media – contains 2% agar
Colony morphology, pigmentation, hemolysis can be
appreciated.
Eg: Nutrient agar, Blood agar
Liquid media – no agar.
o For inoculum preparation, Blood culture, for the isolation of
pathogens from a mixture.
o Earliest liquid medium: urine or meat broth used by Louis Pasteur
Eg: Nutrient broth
Semi solid medium – 0.5% agar.
Eg: Motility medium

13. Solid medium
Semi-solid medium
Liquid
medium

14.  Simple or basal media are culture media which contain the minimum
adequate nutrition for non fastidious organisms
Example:- Nutrient broth/agar
 Most common in routine diagnostic laboratories
Eg: Nutrient Broth, Nutrient Agar
 NB consists of peptone, beeft extract, NaCl, water
• NB + 2% agar = Nutrient agar
• Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium
Uses:-
1. This is basis of most of the media used in the study at common pathogenic
bacteria.
2. It is used for subcultures of certain organisms.
2. BASED ON NUTRITIONAL FACTORS
A. Simple media
Nutrient broth
Nutrient agar

15. B.. COMPLEX MEDIA
 Media that contain some ingredient of unknown chemical composition (exact
chemical composition not known ).
 Complex media may provide complete nutritional requirement of many different
microorganism( fastidious) whose nutritional requirement not known.
 They have added ingredients.
 Provide special nutrients
 It usually contain complex material of biological origin etc blood, milk, peptone,
beef extract, yeast extract.
 Eg; Nutrient broth, Tryptic soya broth, Mac Conkey Agar, soil extract agar, Oat
meal agar

16. C. Synthetic or defined media
Defined media are often used to culture photolithotrophic autotrophs such as
cyanobacteria and photosynthetic protists. They can be grown on relatively simple
media containing CO2 as a carbon source (often added as sodium carbonate or
bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate, and a
variety of minerals. Example :- Dubo’s medium
Media prepared from pure chemical substances and its exact composition is known.
Defined media are used widely in research, as it is often desirable to know what the
experimental microorganism is metabolizing.

17. D. SEMIDEFINED MEDIA
In these media the exact chemical composition of the constituents is not known because
substances like meat and peptone are used. Eg: peptone water – 1% peptone + 0.5% NaCl in
water
Most of the culture media used for routine diagnostic work are semidefined culture
media.
SPECIAL MEDIA (FUNCTIONAL TYPE OF MEDIA
. Enriched media
Substances like blood, serum, egg are
added to the basal medium.
Used to grow bacteria that are
exacting in their nutritional needs.
 Examples:-
Blood agar,
Chocolate agar

18. TYPE : Enriched media.; APPEARANCE : Red color; COMPOSITION :
Sterile Nutrient agar + Defibrinated sheep blood
USES :
 Routine culture
 Widely used in medical bacteriology
 It is also an indicator medium showing the haemolytic
properties of bacteria such as Streptococcus pyogenes.
BLOOD AGAR

19. Choclate Agar Media
 TYPE : Enriched media.; Appearance : Chocolate brown
color.
Melt the desired amount of nutrient agar.
Cool it in a water – bath at 75º C .
Add 10 ml of sterile blood .
Allow the medium to remain at 75º C.
Mixing the blood and agar by gentle agitation from time to time
until the blood become chocolate brown in color, within about
10 min.
USES
 CULTURE OF Neisseria
 CULTURE OF Haemophilus influenzae
 CULTURE OF Pneumococcus
 - to supply blood factors like X factor (Hemin) and Y factor (NAD)

20. Enrichment media
In this media nutritional environment is adjusted such a
manner as to enhance selectivity the growth of certain
bacterial type in a given mix population.eg extract of plant
and animal tissue in NB &NAM
Liquid media used to isolate pathogens from a mixed
culture.
Media is incorporated with inhibitory substances to
suppress the unwanted organism.
Eg: Selenite F Broth – for the isolation of Salmonella, Shigella
–
It is enrichment medium for culture of Salmonella typhi
and paratyphi bacilli from stool sample
Principle:- at neutral pH solution acid salinity has high
toxicity to coli form group of bacteria and not to most
of the salmonella groups.
 Alkaline Peptone Water – for Vibrio cholerae
Selenite F Broth
Alkaline Peptone Water

21. C. Selective media
 It is a medium favor the growth of particular microorganisms in which certain substances are
present which inhibit all other bacteria except the desired bacteria. It encourages the growth of
particular species from a mixed inoculum.
 Bile salts or dyes like basic fuschsin and crystal violet favor the growth of gram-negative bacteria
by inhibiting the growth of gram-positive bacteria; the dyes have no effect on Gram negative
organisms.
 The inhibitory substance is added to a solid media.
 Endo agar, eosin methylene blue agar, and Mac Conkey agar are three media widely used for
the detection of E. coli and related bacteria in water supplies and elsewhere.
Eg:
• Mac Conkey’s medium for Gram negative bacteria
Eosine methylene medium for Gram negative bacteria
• Thiosulfate Citrate Bile Salts Sucrose(TCBS) – for V. cholerae
• Lowenstein Jensen (LJ medium) – M. tuberculosis
• Wilson and Blair medium – S. typhi
• Potassium tellurite medium – Diphtheria bacilli

22. Example:- TCBS
-It is light green translucent medium kept in petridish
-It is selective medium for Vibrio cholera
-Principle:-
Bile salt inhibit the growth of normal
commensals (unwanted bacteria).
Vibrio chloerae produce acid by fermentation
of sucrose which acts on bromothymol blue
(indicator) producing yellow colonies.
Example:- EMB agar media
•selective for gram negative bacteria
•The dye methylene blue in the medium inhibit the growth of gram positive
bacteria
Example:- Lowenstein –Jenson medium
• is solid medium used for Mycobacterium tuberculosis.
• contain penicillin, nalidixic acid and malachite green to inhibit growth of
gram positive and gram negative bacteria,
• in order to limit growth to Mycobacteria species only

23. Potassium Tellurite media
Potassium Tellurite act as selective agent and Inhibit the growth of most
Gram positive and Gram negative bacteria except Corynebacteria diphtheriae
 Used for isolation of Corynebacteria diphtheriae
Reduce Potassium Tellurite to tellurium and produce gray black colour
colony
Direct detection from nose and throat by using swabe
Wilson-Blair medium
contain an indicator which changes its colour when a bacterium grows in them.
The black colour of colonies is due to the ability of these organisms to reduce bismuth
sulphite to sulphide in the presence of glucose
 coliforms are inhibited by brilliant green and bismuth sullphite
For isolation of Salmonella typhi and S. paratyphi
– S. typhi forms black colonie

24. . Differential media
 Differential media are media that distinguish among different groups of microbes
and even permit tentative identification of microorganisms based on their
biological characteristics.
 A media which has substances incorporated in it enabling it to distinguish
between bacteria.
 Blood agar is both a differential medium and an enriched one. It distinguishes
between hemolytic and non-hemolytic bacteria. Hemolytic bacteria (e.g., many
streptococci and staphylococci isolated from throats) produce clear zones around
their colonies because of red blood cell destruction
 Eg: Mac Conkey’s medium (both a differential and selective medium)
 Peptone
 Lactose
 Agar
 Neutral red
 Taurocholate
 MacConkey agar is a culture medium designed to grow Gram-negative bacteria. It
is a useful medium for the cultivation of enterobacteriacea in water and
biological specimens.
 Distinguish between lactose fermenters & non lactose fermenters.

25.  MacConkey agar showing both lactose and non-lactose fermenting colonies.
 Lactose fermenting colonies are pink whereas non-lactose fermenting ones are
colourless or appear same as the medium
It contains lactose and neutral red to distinguish the lactose- fermenting coliforms from the
lactose non –fermenting salmonella and shigella groups.
It contains Bile salts to inhibit non-intestinal bacteria and most Gram-positive bacteria, except
Enterococcus and some species
of Staphylococcus i.e. Staphylococcus aureus.
Neutral red dye : which stains microbes fermenting lactose.
Crystal violet dye : which also inhibits certain Gram-positive bacteria).

26. Thank You

27. Gram-negative bacteria growing on the media are differentiated by their ability to ferment the sugar
lactose.
Lactose fermenter cause the pH to drop and is detected by neutral red, (red at pH's below 6.8.) which
appear as bright pink to red colonies on the agar.
Uses
Acting as a visual pH indicator, the agar distinguishes those Gram-negative bacteria that can ferment
the sugar lactose (Lac+) from those that cannot (Lac-).
 By utilizing the lactose available in the medium, Lac+ bacteria such as
 Escherichia coli
 Enterobacter spp.
 Klebsiella spp.
will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of
red/pink colonies

28.  It is a valuable non-inhibitory growth medium used in
the isolation and differentiation of urinary organisms.
Being electrolyte deficient, it prevents the swarming of Proteus species
(Proteus mirabilis and Proteus vulgaris are frequently involved in urinary
tract infection
Cystine Lactose Electrolyte Deficient medium(CLED
Serretia

29. . Transport media
 Media used for the temporary storage transporting the samples .
 Minimize bacterial outgrowth from time of c
 ollection to time its received at the laboratory to processed.
 Delicate organisms may not survive the time taken for transporting the specimen
without a transport media.
 Such media ideally maintain the viability of all organisms in the specimen without
altering their concentration.
 Transport media typically contain only buffers and salt.
 The lack of carbon, nitrogen, and organic growth factors prevents microbial
multiplication.
 Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent
to prevent oxidation and charcol to neutralize bacterial inhibitor for
gonococci.
Buffered glycerol saline – Used to transport feces suffering from bacillary
dysentery caused by enteric bacilli

30. Anaerobic media
 These media are used to grow anaerobic organisms.
 Semisolid media with reducing agent sodium
thioglyclate and cystine added to provide reduce
environment
 Eg: Robertson’s cooked meat medium,
 Thioglycolate medium.