Microbiology

1. CCULTURE MEDIA USEDULTURE MEDIA USED
IN MICROBIOLOGYIN MICROBIOLOGY
Tuba Bashir
Supervisor Microbiology Department
THE INDUS HOSPITAL, KARACHI

2. Story Behind Penicillin
The discovery of penicillin, one of the world’s first
antibiotics, marks a true turning point in human history
when doctors finally had a tool that could completely
cure their patients of deadly infectious diseases.
Upon examining some colonies of Staphylococcus
aureus, Dr. Fleming noted that a mold called
Penicillium notatum had contaminated his Petri dishes.
After carefully placing the dishes under his microscope,
he was amazed to find that the mold prevented the
normal growth of the staphylococci.

4. Definition, purpose/importance
History of culture media
Classification of culture media
Growth pattern of bacteria
Culture media are vital to microbiology ,
Without good media there is little chance that good
results will emerge from the laboratory

5. Microbiological culture
Method of cultivating microbialMethod of cultivating microbial
organisms by letting themorganisms by letting them
reproduce in predetermined culturereproduce in predetermined culture
media under controlled laboratorymedia under controlled laboratory
conditions.conditions.

6. Bacteria have to be grown (cultured) for themBacteria have to be grown (cultured) for them
to be identified and subsequent clinicalto be identified and subsequent clinical
diagnosis.diagnosis.
By appropriate procedures they have to beBy appropriate procedures they have to be
grown separately (isolated) on culture mediagrown separately (isolated) on culture media
and obtained as pure for study.and obtained as pure for study.
Bacteria have to be cultured in order to obtainBacteria have to be cultured in order to obtain
antigens from developing serological assay forantigens from developing serological assay for
vaccines.vaccines.
Certain genetic studies and manipulations ofCertain genetic studies and manipulations of
the cells also need that bacteria be cultured inthe cells also need that bacteria be cultured in
vitro.vitro.

7. Louis PasteurLouis Pasteur used simple broths madeused simple broths made
up of urine or meat extracts.up of urine or meat extracts.
Robert KochRobert Koch realized the importance ofrealized the importance of
solid media and usedsolid media and used potato piecespotato pieces toto
grow bacteria. It was on the suggestion ofgrow bacteria. It was on the suggestion of
Fannie EilshemiusFannie Eilshemius, wife of, wife of Walther HesseWalther Hesse
(who was an assistant to Robert Koch)(who was an assistant to Robert Koch)
that agar was used to solidify culturethat agar was used to solidify culture
media.media.
History of culture medias

8. Before the use of agar, attempts were made toBefore the use of agar, attempts were made to
use gelatin as solidifying agent.use gelatin as solidifying agent. GelatinGelatin had somehad some
inherent problems….inherent problems….
It existed as liquid at normal incubatingIt existed as liquid at normal incubating
temperatures (35-37temperatures (35-37oo
C)C)
Digested by certain bacteriaDigested by certain bacteria

9. AgarAgar
 Used for preparing solid mediumUsed for preparing solid medium
 Obtained from seaweeds.Obtained from seaweeds.
 No nutritive valueNo nutritive value
 Not affected by the growth of the bacteria.Not affected by the growth of the bacteria.
 Melts at 98Melts at 98oo
C & sets at 42C & sets at 42oo
CC
 2% agar is employed in solid medium2% agar is employed in solid medium
 Agar Fibers Agar PowderAgar Fibers Agar Powder

11. Bacterial Growth Curve

12. Properties of MediaProperties of Media::
• Support the growth of the bacteria.
• Should be nutritive (contains the required
amount of nutrients).
• Suitable pH (neutral to slightly alkaline 7.3-7.4).
• Suitable temperature, and suitable atmosphere.
(Bacteria grow at 370
C)
• Note:Note: media are sterilized by autoclaving at 1210
C and 2
atmosphere for 15-20 minutes. With the autoclave, all
bacteria, fungi, viruses, and spores are destroyed. Some
media can’t be sterilized by autoclaving because they
contain eggs or carbohydrates .

13. Classification:
Bacterial culture media can be
classified in at least three ways
1.CONSISTANCY
2.NUTRITIONAL COMPONENT
3.FUNCTIONAL USE

14. 1) Classification based on
consistency:
A. Liquid media :
.
.
These are available for use in test-tubes, bottles or flasks.
Liquid media are sometimes referred as “broths”
e.g nutrient broth
BHI
In liquid medium, bacteria grow uniformly producing
general turbidity

15. B.Solid media:
An agar plate is a Petri dish that contains a growth mediumAn agar plate is a Petri dish that contains a growth medium
(typically agar plus nutrients) used to culture microorganisms(typically agar plus nutrients) used to culture microorganisms
Agar is the most commonly used solidifying agent

16. C) Semi-solid agar:
Such media are fairly soft and are useful in demonstrating
bacterial motility and separating motile from non-motile
strains
e.g: SIM

17. 2) Classification based on
nutritional component:
Simple media:
Simple media such as peptone water, nutrient agar can support most non-
fastidious bacteria.
Complex media such as blood agar have ingredients whose exact components
are difficult to estimate
Complex media:Complex media:
specially prepared media for research purposes where the composition
of every component is well known.
Synthetic media:Synthetic media:

18. 3) Classification based on functional
use or application:
Basal media
Enriched media
Selective media
Enrichment media
Differential media
Transport media
Anaerobic media
Media for Biochemical Identification

19. 3) Classification based on functional
use or application:
• Basal media
Basal media are basically simple media that supports most non-
fastidious bacteria
Examples of Basal media:Examples of Basal media:
Peptone water,
nutrient broth and
nutrient agar

20. 3) Classification based on functional use or
application:
Enriched mediaEnriched media
contain the nutrients required to support the
growth of a wide variety of organisms, including
some of the more fastidious ones. They are
commonly used to harvest as many different
types of microbes as are present in the specimen.
Examples of Enriched media:Examples of Enriched media:
Blood agarBlood agar
Chocolate agarChocolate agar

21. BLOOD AGAR
• Blood agar plate (BAP) Contains mammalian blood
(usually sheep or horse), typically at a concentration of
5–10%. BAP are enriched, differential media used to
isolate fastidious organisms and detect hemolytic
activity

22. CHOCOLATE AGAR
• Chocolate agar - is a non-selective, enriched growth
medium. containing red blood cells that have been lysed
by slowly heating to 80 °C. Chocolate agar is used for
growing fastidious bacteria, such as Haemophilus
influenzae

24. 3) Classification based on functional
use or application:
Selective media
Any agar media can be made selective by addition of certain inhibitory
agents that don’t affect the pathogen.
To make a medium selective include addition of
antibiotics, dyes, chemicals, alteration of pH or a combination of these

25. • LOWEINSTON JENSON
(L.J )IS A SOLID
MEDIUM USED FOR
MTB
• Sabouraud Dextrose
Agar (SDA)
• Thiosulfate-citrate-bile
salts-sucrose agar
(TCBS)

26. 3) Classification based on functional use or
application:
Enrichment media
liquid media that also serves to inhibit commensal in the clinical specimen.
Selenite F broth and alkaline peptone water are used to recover
pathogens from fecal specimens.

27. 3) Classification based on
functional use or application:
Differential media
Certain media are designed in such a way that different bacteria can be
recognized on the basis of their colony colour
Various approaches include incorporation of dyes, metabolic substrates etc, so that
those bacteria that utilize them appear as differently coloured colonies.
Example of differential mediaExample of differential media
MacConkey’s agar, CLED agar, XLD agar
etc

28. MacConkey Agar
culture medium designed to
grow Gram-negative bacteria
and differentiate them for
lactose fermentation
It contains bile salts (to inhibit
most Gram-positive bacteria),
crystal violet dye (which also
inhibits certain Gram-positive
bacteria)

30. CYSTEINE LACTOSE
ELECTROLYTE DIFFECIENT
AGAR
• C.L.E.D. Agar is a non
selective solid medium for
cultivation of pathogens from
urine specimens. Lack of salts
(electrolytes) inhibits
swarming of Proteus sp.
• With Andrades Indicator

32. BIGGY
Bismuth Sulfite Glucose Glycine Yeast Agar
is a selective and differential medium
used in the isolation and presumptive
identification of Candida spp
Examples of Differential media

34. 3) Classification based on functional
use or application:
Transport media
Clinical specimens must be transported to the laboratory
immediately after collection to prevent overgrowth of contaminating
organisms or commensals. This can be
achieved by using transport media.
Example of Transport mediaExample of Transport media
Cary Blair medium for campylobacter species
Alkaline peptone water medium for V. cholerae

35. Some important criteria of
Transport media
Transport media should fulfill the following criteria:
• temporary storage of specimens being transported to the laboratory
for cultivation.
• maintain the viability of all organisms in the specimen without
altering their concentration.
• contain only buffers and salt.
• lack of carbon, nitrogen, and organic growth factors so as to prevent
microbial multiplication.
• transport media used in the isolation of anaerobes must be free of
molecular oxygen.
• Transport Swab features a 5 mL Amies agar gel column with oxygen-
scavenging agents, for sampling of both aerobic and facultative anaerobic
organisms.

36. 3) Classification based on functional
use or application:
Anaerobic media
Anaerobic bacteria need special media for growth because they need
low oxygen content, reduced oxidation –reduction potential and extra
nutrients.
Media for anaerobes may have to be supplemented with nutrients like
hemin and vitamin K
Example of Anaerobic mediaExample of Anaerobic media
Thioglycollate medium

38. Media for Biochemical
Identification
TSI
Urease
Simmon’s Citrate
SIM

41. Mueller-Hinton agar
it is a non-selective, non-differential
medium
it contains starch. Starch is known to absorb
toxins released from bacteria, so that they
cannot interfere with the antibiotics
it is a loose agar. This allows for better
diffusion of the antibiotics than most other
plates. A better diffusion leads to a true
zone of inhibition.

43. Quality tests
chemical and biological parameter
checked to ensure end products
meet product quality specification,
packaging,labelling and storage are
important