7_CULTURE_MEDIA.pptcvvc f

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1. WELCOME TO
CULTURE MEDIA

2. CULTURE MEDIA
Introduction 
For good identification and valid laboratory results, there
is need of broad selection of media for culture of micro-
organisms.
For culture media to be successful in growing micro-
organisms it must have the following nutrients: ions,
moisture, maintain the correct PH, osmotic pressure
and neutralize any toxic materials produced by the
growing organism.
The grown organisms should be
incubated in the correct atmosphere,
at the optimum temperature and for
an adequate period of time.

3. CLASSIFICATIONS OF MEDIA
Culture media is classified into (5) five classes depending
entirely on:
The ingredients the media contains
What types of micro-organisms are capable of growing
in it?
The five classes of media are;
Basic media
Enriched and enrichment media
Selective media (inhibitory media)
Differential (indicator media)
Transport media
Identification media

4. 1. BASIC MEDIA
Definition:
These are simple culture media, which can be in solid or liquid
form. They contain the basic nutritional requirements for
the
bacterial growth.
use
They are used in:
Maintenance of micro-organisms as control stains
Performing biochemical and serological tests
Setting of drug susceptibility testing
Sub-culturing micro-organisms for selective and
differential media
Preparation of enriched media
Examples: Nutrient agar medium, Nutrient broth etc.

5. 2. ENRICHED AND ENRICHMENT MEDIA
Enriched
Definition
These are media in solid form and are enriched with the basic
nutritional requirement for the bacterial growth such as whole
blood, lysed blood, peptones, vitamins, serum and special meat
extract.
Uses
It is used in growth of fastidious organisms (e.g.
Haemophillus influenzae, Neisseria spps, Streptococcus spps, TB,
Corynebacterium diphtheriae) that requires extra nutritional
growth requirements.
Examples:
Blood agar medium (BA)
Sheep blood agar medium (SBA)
Chocolate blood agar media (CBA)

6. Cystine lactose electrolyte deficiency medium (CLED)
Lowenstein Jensen medium (T.B)
Loeffler's media (C. diptheriae)
Robertson cooked meat medium (anaerobic organisms)
Enrichment media
Definition
These are media in broth or liquid form. The medium
favors or suppresses growth of some micro-organism
due to the production of toxins in the media.
Uses
Used for culturing micro-organisms that are to be sub-
cultured to selective media e.g. DCA, XLD, SS, TCBS,
Monsures medium etc.

7. Examples
Selenite F broth for Salmonella and Shigella spps.
Alkaline peptones water broth for V. cholera, Y. Pestis
Tetrathionate broth for Salmonella and Shigella spps.
3. SELECTIVE MEDIUM (INHIBITORY MEDIA)
Definition
These are media in solid form which contains inhibitory
substances such as:
Bile salts e.g. Sodium tourochollate, Sodium
deoxychollate
Citrate salts e.g. Sodium citrate
Antibiotics
pH etc.

8. Uses
Used to inhibit growth of organisms other than the
microorganisms in which the media is designed for.
Used for culturing specimen from a site with normal floras
to prevent growth of unwanted micro –organisms.
Examples
DCA (Deoxychollate Citrate Agar) for Salmonella and
Shigella Spps.
SS (Salmonella/Shigella) media for Salmonella/Shigella
Spps
TCBS (Thiosulphate citrate bile salts) for V. cholera
Buztler media for Campylobacter spps.
–Xylose lysine deoxychollate agar (XLD) for Salmonella/
Shigella Spps

9. 4. DIFFERENTIAL MEDIA (INDICATOR MEDIA)
Definition
These are media which contains substances such as indicators,
dyes or any other changeable substances that can be visibly
changed as a result of metabolic activities of a particular micro-
organism.
The effect of the metabolic activities of these micro-organisms
can be shown with an indicator (change of color) or blood cells
reaction (hemolysis).
Examples
MacConkey medium:
contain indicator (neutral red) which changes to pink after
fermentation of carbohydrates (Lactose)
Differentiates lactose fermenters (E. coli, Klebsiella spps from
non-lactose fermenters (Pseudo. spps, proteus spps).

10. SBA
Differentiate hemolytic organisms from non-hemolytic organisms
e.g. Streptococcus pyogenes.
TCBS
Has an indicator (Bromothymol blue) which changes to yellow
after fermentation of sugar (sucrose)
Differentiates sucrose fermenters from non –sucrose fermenters
e.g. V. cholerae



5. TRANSPORT MEDIA
Definition
These are usually semi-solid media which contains ingredients
that prevents growth of commensal and ensures survival of
aerobic and anaerobic pathogenic micro-organisms when
specimen cannot be cultured immediately or soon after collection.

11. Uses
Used in transportation of micro-biological specimen for
health centers to the district or provincial microbiology
benches.
Examples
Cary Blair media (non nutrient soft agar gel containing a
reducing agent ) for V. cholerae, Y. pestis
Stuarts media (armies transport media) for Neisseria Spps

12. PREPARATION OF SPECIFIC CULTURE MEDIA
Medium :Medium : medium is a nutrient blend (component) used to
support microbial growth (growth of micro-organisms).
There are three physical forms of media, broth, solid, and
semisolid.
1. Solid media1. Solid media:
Are prepared by adding a solidifying agent (1.5～3.0% of agar).
Prepared mainly in Petri dishes, but also in tubes and slopes. After
growth the bacterial colonies are visible. e.g. blood agar,
chocolate agar, MacConkey agar.
They promote surface growth of micro-organisms.
They are used to isolate pure cultures
They are ideal for culture storage
They are helpful in the observation of biochemical reactions
They are used to make slants, deeps, and plates (named by
medium)

13. 2. Semisolid agar2. Semisolid agar (soft agar):
Contains small amounts of agar (0.3～0.7% of agar)
Used to check for motility
Used as a transport media for fragile organisms.
Can have semisolid agar in Petri dishes or in tubes. In tubes
it is usually slanted to increase surface area.
3. Liquid (Broth):3. Liquid (Broth):
Mostly used for biochemical tests (blood culture, Broth
culture).
Growth of bacteria is shown by turbidity in medium. e.g.
Nutrient
broth, Selenite F broth, alkaline peptone water.
surface growth pellicleuniformly turbidsediment in bottom

14. Properties of agar:
Some what like gelatin.
It melts at 97
0
C and solidifies at 37
0
C.
Comes as solid powder and then you add water to it.
Properties of Media:
Support the growth of the bacteria.
Should contains the required amount of nutrients for
bacterial growth.
Should have a suitable pH (neutral to slightly alkaline 7.3-
7.4).
Should have a suitable temperature, and suitable
atmosphere. (Bacteria grow at 37
0
C)
NoteNote:: media are sterilized by autoclaving at 121
0
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 .

16. Clinical Microbiology Laboratory Bench

17. 1. NUTRIENT AGAR
This is a solid agar medium that is used for isolation of
colonies
e.g. when doing bacterial cell counts, description of colonies
Morphology e.g. Meat extract broth, Digest broth etc.
Ingredients
•Peptones
•Nutrients agar powder
•Yeast extract
•Sodium chloride
•Distilled water
Preparation
Weigh 28gms of Nutrient agar powder in 1000mls of distilled
water (Follow manufacturers instructions).

18. Cap the flasks and Sterilize by autoclaving at 121
0
c and
pressure (15 psi) for 15-20minutes.
Aseptically pour or dispense (15-20mls) of the molten
Nutrient agar broth into culture plates and let it solidify as it
cools.

20. Label culture tubes or Petri plates using a marker with the
Initials, Date (mm/dd/yy) and the Code number.
store the plates at 2-3
0
c in a plastic bag to prevent loss of
moisture and also upside down to prevent condensation.
Shelf life – up to 2 years
PH. Should be (PH 7.2-7.6) at room temperature

22. 2. BLOOD AGAR MEDIUM
This is an enriched medium which is used for growth of
organisms
which are too fastidious to growth in nutrients Agar medium. It
is
Also used in demonstration of blood cell lysis (haemolysis)
Example
Sheep blood agar medium (SBA): good for showing haemolysis
Rabbit blood agar medium (RBA): good for providing V-factors
for growth of Haemophillus influenzae organism
Horse blood agar medium (HBA): its cheap and free from
antibiotics
Human blood agar medium (HBA): very cheap to prepare and
easily available
Disadvantages: may contain some antibacterial substances.

23. Ingredients
Beef heart: source of nutrients
Tryptose: source of nutrients
Sodium chlorides: source of salts
Agar: solidifying agents
Distilled water: solvent, blood
Preparation
Prepare the agar medium as per the manufacturers
instructions (dissolve 37.0gms of Blood agar powder base in
1000mls of distilled water)
Cap the flasks and Sterilize by autoclaving at 121
0
c and
pressure (15 psi) for 15-20minutes.
Cool the molten agar in a flask with water up to 50
0
c.
 Add 5-10% whole blood (5-10mls) into 100mls of molten
agar cooled at 50
0
c

24. Aseptically pour or dispense (15-20mls) of the molten Blood
agar broth into culture plates and let it solidify as it cools.
Label culture tubes or Petri plates using a marker with the
Initials, Date (mm/dd/yy) and the Code number.
Store the plates at 2-8
0
c in a plastic bag to prevent loss of
moisture and also upside down to prevent condensation.
Test for sterility and potency of the prepared medium
Sterility: incubate the medium overnight and check for
growth of any contaminants
Potency: inoculate organisms specific for that medium and
check whether they grow

25. 3. CHOCOLATE BLOOD AGAR MEDIA (CBA)
This is an enriched medium for growth of fastidious organism
such as Haemophillus influenzae, Streptococcus pneumoniae,
Neisseria spps.
Ingredients
Same as for Blood agar medium
Preparation
Prepare the agar medium as per the manufacturers
instructions (dissolve 37.0gms of Blood agar powder base in
1000mls of distilled water)
Cap the flasks and Sterilize by autoclaving at 121
0
c and
pressure (15 psi) for 15-20minutes.
Cool the molten agar in a flask with water up to 80
0
c.
 Add 5-10% whole blood (50-100mls) into 1000mls of molten
agar, cook until it turns chocolate brown or cook for 10-15min.

26. Aseptically pour or dispense (15-20mls) into culture plates
(petri dishes) and let it solidify as it cools.
Label culture plates (Petri dishes) with the Initials, Date
(mm/dd/yy) and the Code number using a marker
Store the plates at 2-8
0
c in a plastic bag to prevent loss of
moisture and also upside down to prevent condensation.
Test for sterility and potency of the prepared medium
Sterility: incubate the medium overnight and check for
growth of any contaminants
Potency: inoculate organisms specific for that medium
and check whether they grow

27. 4. THAYER MARTIN MEDIUM (MODIFIED CBA)
This is chocolate blood agar medium added drugs such as;
Vancomycin: inhibits growth of gram +ve organisms
Colistin: inhibits growth of gram –ve organisms
Nystatin: inhibits growth of fungi
This medium is important for the isolation of Neisseria
gonorrheae, Neisseria meningitides and Haemophillus
influenzae
 
The specimen are: throat swabs, vaginal swabs

28. 5. MACCONKEY AGAR MEDIUM
It is both a selective & differential media.
It is a selective media because it inhibits growth of some
organisms [Gram positive bacteria].
It is a differential medium for lactose and non- lactose
fermenters organisms especially enteric
organism ;contains neutral red indicator which turns to
pink upon fermentation of lactose sugar (organisms in
the alimentary canal)
No lactose fermentation Lactose fermentation
MacConkey Agar

29. INGREDIENTS
Peptones: source of nutrients
Agar: solidifying agent
Bile salts e.g. Sodium taurochollate, Sodium deoxychollate
(which inhibits growth of gram +ve organisms)
Indicators: Neutral red which is red in alkaline medium and
pink in acid medium.
Carbohydrates such as Lactose: fermentable sugars
Distilled water: as a solvent
PREPARATION
Prepare the agar medium as per the manufacturers
instructions (dissolve 52gms of MacConkey powder into
1000ml of distilled water).
Cap the flasks and Sterilize by autoclaving at 121
0
c and
pressure (15 psi) for 15-20minutes.

30. Cool the medium up to (50-55
0
c) and dispense 15mls
aseptically into sterile Petri-dishes
Label culture plates (Petri dishes) with the Initials, Date
(mm/dd/yy) and the Code number using a marker
Store the plates at 2-8
0
c in a plastic bag to prevent loss of
moisture and also upside down to prevent condensation.
Test for sterility and potency of the prepared medium
Sterility: incubate the medium overnight and check for
growth of any contaminants
Potency: inoculate organisms specific for that medium
and check whether they grow
Shelf life is up to 4 weeks

31. Expected results
Lactose fermenters e.g. E. coli, Enterococci, Kleb.
pneumoniae will have pink colonies due to fermentation
of lactose
Non-lactose fermenters e.g. Shigella, Salmonella, Proteus,
Pseudomonas aeroginosa, Y. enterocolitica produces red
colonies since no fermentation of lactose
Specimen
Stool specimen
Urine specimen
Water sampling
Pathogens in stool
Salmonella spps, Shigella spps, Vibrio, Escherichia spps for under
5yrs, Staph. aureus, Clostridium botulinum etc.

32. 6. C.L.E.D (Cystine Lactose Electrolytes Defficient) Media
It is a media of choice for urine specimen
It is a differential media which differentiates lactose
fermenters from non-lactose fermenters
Its electrolytes deficiency prevents swarming of proteus spps
It is not a selective media because any organism isolated
in it is of vital importance
Ingredients
Agar: solidifying agent
Cystine: source of nutrients
Meat extract: source of nutrients
Lactose: fermentable sugar
Bromothymol blue: an indicator that is blue in alkaline
medium and yellow in acidic medium.
Peptones: sources of nutrients
Tryptone: source of nutrients

33. Nb. The medium does not have salts hence prevents proteus
spps
from swarming
Preparation
Prepare the medium as per the manufacturers instructions
(dissolve 36.2gms of commercially available CLED medium
powder into 1000mls of distilled water)
Cap the flasks and Sterilize by autoclaving at 121
0
c and
pressure (15 psi) for 15-20minutes.
Cool the medium up to (50-55
0
c) and dispense 15mls
aseptically into sterile Petri-dishes
Label culture plates (Petri dishes) with the Initials, Date
(mm/dd/yy) and the Code number using a marker
Store the plates at 2-8
0
c in a plastic bag to prevent loss of
moisture and also upside down to prevent condensation.

34. Test for sterility and potency of the prepared medium
Sterility: incubate the medium overnight and check for
growth of any contaminants
Potency: inoculate organisms specific for that medium
and check whether they grow
(shelf life -4week)
Expected results
Lactose fermenters e.g. E. coli, Klebsiella, Staph. Aureus,
Strep. pyogenes will produce yellow colonies
Non-lactose fermenters e.g. Shigella, Salmonella, Proteus
spps will produce colorless (pale blue color) colonies.
Pathogens in urine
Escherichia coli, Staphylococcus aureus, Streptococcus faecalis,
Proteus spps, Klebsiella spps, Candida albican

35. 7. D.C.A (Deoxycholate Citrate Agar) Medium
It is a selective media for Salmonella and Shigella Spps
It is also a differential medium that differentiates lactose
fermenters from non-lactose fermenter organisms
It also isolates Yersinia spps when extra bile salts are added.
Ingredients
Agar: solidifying agent
Peptones: source of nutrients
Distilled water: solvent
Neutral red: indicator (red in alkaline, pink in acidic
Lactose: fermentable sugar
Bile salts
Sodium deoxychollate: inhibits growth of gram+ve organisms
Sodium citrate: inhibits growth of organisms like E. coli, Klebsiella
Spps

36. Sodium thiosulphate: inhibits growth of organism like E.
coli, Klebsiella and Proteus spps.
Ions salts e.g.
Ferric citrate: reacts with hydrogen sulphide gas to
produce a black precipitate (H
2
S). Its work is to neutralize
toxins produced by the growing organisms to allow
growth of Salmonella and Shigella spps
PREPARATION
Prepare the agar medium as per the manufacturers
instructions (dissolve 52gms of commercially available DCA
powder into 1000ml of distilled water).
The other steps are as for preparation of CLED agar
medium.
Shelf life is 6 weeks

37. Expected results
Lactose fermenters e.g. E. coli, Klebsiella spps produces pink
colonies
Non-lactose fermenters e.g. Salmo. /Shigella spps colourless
colonies
Salmonella spps produces colorless colonies with
blackening at the center due to H
2S production.
Nb
Do not autoclave the medium because autoclaving destroys
ions
salts e.g. Sodium citrate, sodium thiosulphate.

38. 8. SELENITE F (FAECES) BROTH
This is an enrichment medium used for isolation of enteric
pathogens (bacteria in the elementary canal)
Chemical- like substances produced by the bacteria kills
most of the other bacteria growing in the media except the
pathogens that the medium is intended for.
The system of killing stops after (18-24hrs) and therefore
sub –culture must be done.
It is a medium for salmonella Spps.
Ingredients
Sodium acid Selenite: it is a toxin that prevent growth of E.
coli
Peptone: source of nutrients
Mannital: fermentable sugar
Di-sodium hydrogen phosphate: as a buffer

39. Nb:
After 8-12 hrs in the broth, sub-culture into a solid medium
of choice i.e. DCA or SS medium
Store the broth at room temperature

40. 9. TETRATHIANATE BROTH
It is a selective enrichment broth used for recovery of
Salmonella spps.
Ingredients
Peptones base with yeast extract
Mannital glucose, calcium carbonate
Brilliant green that inhabits growth of gram + ve
Sodium deoxychollate and gram –ve organisms other
than
Sodium thiosulphate Salmonella Spps
After 8-12hrs sub-culture in a solid medium
Store at room temperature

41. 10. SALMONELLA /SHIGELLA (SS) AGAR MEDIUM
This is a highly selective medium for the recovery of Salmonella
and Shigella spps
Ingredients
Beef extract
Bile salts
•Sodium thiosulphate inhibits growth of +ve
•Sodium deoxycholate organism
•Sodium citrate inhibits growth of gram –ve
•Brilliant green organism
Peptones
Lactose
Neutral red which is red in alkaline and pink in acid media
Ferric salts: its metabolism produces H
2
S

42. Preparation
Prepare the medium following the manufacturers
instructions
Dissolve the powder into 1000mls of distilled water
Heat to dissolve and sterilize but do not auto-clave
Let it cool up to (50-55)
0
c and dispense 15-20mls into sterile
Petri-dishes aseptically
Let it cool as it solidifies
Test for its sterility and potency
Label the medium with the date and name of the medium
Put in a plastic bag and store at 2-8
0
c in a refrigerator
Organisms
Salmonellas spps: produces colorless colonies (red colonies)
with black centers
Shigella spps: produces red colorless without blackening

43. 11. XLD (XYLOSE LYSINE DEOXYCHOLATE)
It is both a selective & differential media.
It is a selective media because it inhibits growth of some
organisms [Gram positive bacteria].
It is a differential medium for Sucrose and non-sucrose
fermenters organisms especially enteric organism (organisms
in the alimentary canal)
Ingredients
Agar: solidifying agent
Sucrose and lactose: fermentable sugars Xylose
L- lysine yeast extract salts (ammonium salt)
Sodium deoxycholate inhibits contaminating Gram-positive
flora
Sodium thiosulphate, Sodium chloride
Ferric ammonium sulphate
Distilled water, phenol red, PH 7.2,

44. Preparation
Prepare the medium following the manufacturers
instructions
(dissolve 53.5gms of XLD powder into 1000mls of distilled
water)
Heat to dissolve and sterilize for 15-20 minutes (never
autoclave because autoclaving destroys all bile salts e.g.
sodium deoxycholate
Let it cool up to 50-55
0
c and pour 15-20mls into sterile Petri-
dishes aseptically
Let it cool as it solidifies
Label the plates with the name of the media and the date
the media was prepared
Put it in a plastic bags and store at 2-8
0
c in a refrigerator
Test for the sterility and potency of the media

45. Expected reactions
Shigella and Providencia : produces red color colonies
(NSF)
Salmonella, Arizona, Proteus, Edwardsiella: produces red
color (NSF) colonies with blackening at the center
E. coli, Klebsiella spps, Enterobacter, Citrobactor, Hafnia:
produces yellow color colonies (SF)

46. 12. LOWENSTEN –JENSEN MEDIA (LJ)
This is a complex media for isolation of Mycobacterium
tuberculosis of human type
Ingredients
Beaten egg-York: source of nutrients for M. tuberculosis
Malachite green: inhibits growth of contaminants other
than T.B
Glycerol: provides carbon for growth of M. tuberculosis
Asparagines: provides nitrogen for growth of M. tuberculosis
Mineral salts e.g. magnesium salts, phosphates, sulphates,
citrates
Distilled water

47. Preparation
Solution A
Dissolve mineral salts and Asparagines in 600mls of dist.
Water
Autoclave at 121
0
c for 25minutes
Solution B
Dissolve 2gms of malachite green powder into 100mls of
distilled water
Incubate at 37
0
c for 1-2 hrs
Mix solution A and solution B and make the volume up to
1000mls with beaten egg York.
Dispense 15-25mls of mixture into sterile Bijou bottles
aseptically

48. Organisms
M. tuberculosis (human type) raised dry, wrinkled, buffy
colored (yellow) or white colonies
M. tuberculosis (bovine type) low, smooth, moist, small
colonies

49. 13. THIOSULPHATE CITRATE BILE SALTS (TCBS)MEDIUM
Thiosulfate-Citrate-bile Salts-Sucrose agar or TCBS agar is a type of
selective agar medium for isolation of Vibrio spps which grow well at
35-37°C on media containing 1% Sodium chloride and a very high
pH (8.5-9.5).
Halophilic vibrios require sodium chloride for optimum growth and
metabolic activity.
Ingredients
Sodium citrate
Sodium thiosulphate inhibits growth of commensals
Ox-bile
Yeast extract, peptones, sucrose, ferric citrate
Bromothymol blue (indicator)
1.0% Sodium Chloride
Agar.................1.5%

50. Principle :
TCBS Agar contains a complementary source of plant and
animal proteins, 1% Sodium Chloride, Sodium thiosulphate
source of sulfur (in combination with Ferric citrate, detects
hydrogen sulphide production)., and yeast extract, all of
which allow optimum growth. The Bile Salts in the media
inhibit the growth of gram-positive microbes.
The presence of Sucrose allows for the differentiation of
those vibrios, which can utilize sucrose with the aid of
Bromthymol blue, as a pH indicators.
The high pH (8.5-9.5) of TCBS Agar suppresses other
intestinal flora while allowing uninhibited growth of vibrios.

51. Preparation
Prepare the medium flowing the manufacturers
instructions
Heat the preparation to dissolve and sterilize (do not auto-
clave or overheat because this will destroy bile salts)
Let it cool up to (50-55)
0
c and dispense (15-20)mls into
sterile Petri-dishes aseptically
Let it cool as it solidifies
Label the culture plate with the name of the medium and
date it was prepared
Store the medium in plastic bags at (2-8)
0
c in a refrigerator
Test for its sterility and potency
Shelf life 4weeks

52. Organisms
Vibrio cholerae produces yellow colonies due to
fermentation of sucrose with acid production and
transparent edges
Vibrio para-haemolyticus do not ferment sucrose hence the
colonies are blue-green in color.

53. 14. TSI (TRIPLE SUGAR IRON) MEDIUM
This is a highly differential media for identification of gram
–ve rod (bacilli).
It is a composite media used to study different properties of
a bacterium – sugar fermentation, gas production and H
2
S
production.
In addition to peptone, yeast extract & agar, it contains 3
sugars – Glucose, Lactose, Sucrose.
The Iron salt – Ferric citrate indicates H
2
S production.
Ingredients
Agar: solidifying agent
Peptones: source of nutrients
Iron salts: neutralizes toxins hence allowing growth of gram
–ve bacilli

54. Phenol red: indicator that turns Yellow in acidic media
Sodium thiosulphate: inhibits growth of gram +ve bacilli
Sugar:
Glucose 0.1gms
Lactose 1gm
Sucrose 1gm
Distilled water 1000mls
pH of the medium – 7.4
It is an orange red medium with a slant and a butt.
 

55. Preparation
Dissolve the above ingredients in distilled water following
the manufacturer’s instructions
Boil to dissolve and sterilize (never auto-clave because
autoclaving destroys bile salts e.g. sodium thiosulphate
hence allowing growth of gram +ve organisms)
Cool up to 50-55
0
c and dispense 15-20mls aseptically into
culture tubes and slant the tube as the media solidifies
Test its sterility and potency
Store at room temperature.

56. Principle
•The reaction is based on fermentation of glucose, lactose
and sucrose with production of gas, acid and hydrogen
sulphite depending on the organism involved in the
reaction.
•The organisms involved uses glucose which is in small
quantities thus turning the media from red to yellow.
Oxygen diffuses into the media hence neutralizing the acid
produced thus changing the media back to red. (this is
referred to as oxidation)
•If this media is further left for more than the required time,
it will complete change to red.
•If the growing organisms has the ability to ferment lactose,
sucrose and glucose the media changes to yellow (acidic)
and in the same time gas and H
2
S will be produced
depending on the organism isolated

57. PROCEDURE
Sterilize the inoculating straight wire in the blue flame of
the bunsen burner until it is red hot and then allow it to
cool.
Sterilize the neck and mouth of tube containing the 24-48
hour culture, using aseptic technique, take the culture of
the organism using a straight wire .
Take a sterile TSI slant tube from the rack, remove the cap
and flame the neck of the tube.
Stab the straight wire containing the pure culture into the
medium, upto the butt of the TSI tube, and then streak the
needle back and forth along the surface of the slant.
Again flame the neck of the TSI tube, cap it and place it in
the test tube rack.
Incubate at 37
o
c for 18 to 24 hours.

58. Expected Results:
Yellow – Acid production
Pink - Alkaline or no acid production
Yellow slant / Yellow butt (Acid/Acid) – Lactose fermenters
e.g.
E.coli, Klebsiella
Pink slant / Yellow butt (Alkaline/Acid)– Non lactose
fermenters
e.g. Salmonella, Shigella
Pink slant / Pink butt (Alkaline/Alkaline) – No carbohydrates

fermentation
Black colour – H
2
S production e.g. Proteus
Gas bubbles or crack in the medium – gas production.

59. 1.Alkaline slant (red) and acid butt (yellow) with or
without gas production (breaks in the agar butt):
Only glucose fermentation has occurred.
The organisms preferentially degrade glucose first. Since this
substrate is present in minimal concentration, the small
amount of the acid produced on the slant surface is
oxidized rapidly.
The peptones in the medium are also used in the
production of alkali. At the butt, the acid reaction is
maintained because of the reduced oxygen tension
and slower growth of the organisms. 

60. 2. Acid slant (yellow) and acid butt (yellow) with or without
gas production:
Lactose or sucrose fermentation has occurred.
Since these substances are present in higher
concentrations, they serve as substrates for
continued fermentative activities with maintenance
of an acid reaction in both the slant and the butt. 
3. Alkaline slant (red) and alkaline butt (red)
No carbohydrate fermentation has occurred.
Instead; peptones are catabolized under anaerobic
and aerobic conditions resulting in alkaline pH due to
production of ammonia. If there is aerobic and
anaerobic utilization of peptone, the alkaline reaction
is present on the slant and the butt. 

61. 4.Hydrogen sulfide (H
2
S) production:

Some bacteria utilize Thiosulfate anion as a terminal electron
acceptor, reducing it to sulfide. If this occurs, the newly
-formed hydrogen sulfide   (H
2S) reacts with ferrous
sulfate in the medium to form ferrous sulfide, which is
visible as a black precipitate. The blackening of the
medium is almost always observed in the butt (bottom)
of the medium
5. Carbon dioxide (CO
2) production:

It is recognized simply as air bubbles between the agar
And the wall of the tube or within the agar itself within
18-24 hours following incubation. The carbon dioxide
production is sufficient to split the agar into two or
more sections.

62. Fermentati
on
No
fermentat
of
carbohydr
ates
Fermentati
of glucose
only
Fermentati
of glucose
with gas
production
Fermentati
of glucose
with H
2
­
S
Fermentati
of glucose
with H
2
­
S
and gas
Fermentati
of glucose,
lactose and
sucrose
with gas
Fermentati
of glucose,
lactose and
sucrose
without
gas and H
2
­
S
Fermentati
of glucose,
lactose and
sucrose
with H
2
­
S
production
.
SLANT Alkaline Alkaline Alkaline Alkaline Acid Acid Acid Acid
BUTT AlkalineAcid Acid Acid Acid Acid Acid Acid
Production
of gas
O O + O + + O O
H
2
­
S
Production
O O O + + O O +
Organism *ps.
Aeroginosa
*ps. nimae,
*strep.
faecalis
*
salm.para-
ty
* shigella
* pro.
rettigen
*providenal
e
*salmo-
para
* shigella
*proteu
*salm.typhi
*p.morgani
s
*Arizona
*salmo
typhi
*p.mirabilu
s
*Klebsiella
*salmo.
paratyhi
E. coli
*proteus
Spps
*strept.
spps
*staphyl. sp
*E. coli
*Enterobact
or
*citrobacto
*Arizona

DIAGRAM

64. 15. KLIGLER IRON AGAR (KIA) MEDIA
•This medium is same as TSI except that in the ingredients,
there is absence of sucrose. The reaction is the same except
that there is fermentation of glucose, lactose with
production of H
2
S. It is a low differential media for
identification of Salmonella, Shigella and other enteric
bacteria (in the elementary canal)
 
QUALITY CONTROL OF MEDIA
Culture media should be prepared correctly using
manufacturers instructions (preparation manuals)
Sterility of the culture media should be done by incubating
the medias at 37
0
c overnight (18-24)hrs
Newly prepared media should be tested for its potency by
inoculating and incubating standard organisms at 37
0
c
overnight (18-24) hrs
Culture media should be labeled clearly

65. Culture media should be transported properly to the
districts lab. By packing them well to avoid moisture, direct
sunlight and excessive heat.
Proper storage: culture media should be stored at 2-8
0
c in a
refrigerator in a plastic bags in order to maintain moisture.
Slope media e.g. P
3
broth can be stored at room
temperature.
The expiring date of agar powder should be noted.

66. TOXINS
Definition
These are poisonous substances produced by micro-
organisms during their feeding time. These toxins are
produced by living or dead organisms in their bacterial
multiplication. Toxins are injurious to body tissues and they
are capable of causing diseases.
Virulence of toxins differs from one species of micro-
organism to another species. The chemical structures of
toxins are unknown. They are usually complex organic
compounds whose nature resembles that of proteins.
There are two types of toxins. These are;
Exotoxins
Endotoxins

67. DIFFERENCES BETWEEN EXOTOXINS AND ENDOTOXINS
EXOTOXINS ENDOTOXINS
1Produced by living organisms
and diffuse freely to the
surrounding media
Produced after or at death of micro-
organisms
2Produced by gram +ve
organism
Produced by gram –ve organisms
3Highly antigenic Weakly antigens
4Toxic protein substances which
are specific in their action
Toxic protein polysaccharide lipid
complex, which are non-specific in their
action.
5Can be converted into toxoids Cannot be converted into toxoid
6Are extra-cellular Are intra-cellular
7Are completely neutralized by
specific antibodies to the
bacteria (coming into contact
with the bacteria
Are partially neutralized by specific
antibodies against the bacteria
8Are heat and chemical labile
(sensitive)
Are heat and chemical stable
(resistance)
9Are affected by variety of
chemical agents
(reagents)which denature
toxin
Their complex molecules can be broken
down by phenol extraction which
separates lipo-polysacharides from
protein

68. SPECIFIC TOXINS
DIPTHERIA TOXINS
These are exotoxins, which have the ability to cause injuries in
the
kidney nervous systems and hard tissue.
NECRO- TOXINS
These are toxins which have the ability to injure tissue
causingnecrosis
TETANUS TOXINS
These are toxins produced by organisms called clostridium
tetani
and have the ability to destroy certain nerves. There are
responsible of lock –jaw.

69. STREPTOLYSIN ‘O’
These are produced by Beta Streptococcus pyogenes and are
responsible for destruction of blood cells.
LEUCOCIDIN
They are produced by Staphylococcus organisms and are
responsible for destruction of white blood cells (leucocytes)
HAEMOLYSIN
They are produced by Staphylococcus organisms and other
Organisms and are responsible for destruction of red blood
cells.
ENTERO-TOXINS
They are produced by food poisoning Staphylococcus and are
characterized by diarrhea and vomiting.

70. NEURO-TOXIN
These are poisons that affects nervous system
NB: When toxins are rendered non-poisonous by heat or
chemicals, it is called toxoid.
 
COMMON SYMPTOMS
Toxaemia: This is presence of toxin in blood.
Pyrexia: This is raise in temperature
Prostration: This is general body weakness and fainting.
Haemorphage and aodema : This is characterized by internal
bleeding
 
TOXOIDS
These are toxins which have lost toxicity but have certain
antigenicity .The toxoids are antigenic and toxigenic (capable of
making antibiotics) 

71. ANTI-TOXINS
These are antibodies that are used to neutralize harmful
effects of
toxins. Antitoxins are prepared by immunizing animal with
toxins
or toxoids