isolation of bacteria

1. ISOLATION OF BACTERIAL PATHOGENS
IN PURE CULTURES
(Bacterial Culture media)
Dr Mostafa Mahmoud Ahmed, Ph D
Consultant Microbiologist, GDHA, Riyadh, KSA.
Associate Prof. of Microbiology & Immunology.
Faculty of Medicine – Ain Shams University

2. CULTURE MEDIA
Culture media generally provide sources
of carbon , energy and nitrogen in the form
of available carbohydrates and amino
acids
.
Special media provide specific requirements
as inorganic salts or particular growth
factors
.

3. Types of Culture Media
- Basic media
- Enriched media
- Selective media
- Enrichement media
- Indicator ( Differential ) media
- Transport media

4. I . BASIC MEDIA
- These are simple media used to support the
growth of microorganisms that do not have
special nutritional requirements.
- They include nutrient broth, peptone water,
and nutrient agar.

5. 1
-
Nutrient Broth
-
Filtrate of cooked fresh minced meat + 1%
peptone + 0.5% NaCl
.
-
Clear yellowish fluid medium
.
-
Sterilized in autoclave at
121°C for 30 min
.
-
Base for most culture media
.

6. 2
-
Peptone water
-
1%
peptone + 0.5% NaCl dissolved in
water
-
Clear colourless fluid medium
-
Sterilized in autoclave at 121°C
for 30 min
.
-
Base for sugar media
-
Indole production test

7. 3
-
Nutrient agar
-
2-3%
agar powder dissolved in nutrient
broth
.
-
Yellowish semi-transparent medium
(
Plates or tubes as slopes or deep agar
).
-
Sterilized in autoclave at 121°C for 30 min
.
Uses
:
-
Base of different culture media
-
Plates : for isolation & identification of bacteria
-
Slopes : for preservation of pure cultures
-
Deep agar : for anaerobic bacteria

8. Nutrient Agar Plates

9. II. ENRICHED MEDIA
- Prepared by the addition of substances such
as blood, serum or egg to a basic medium.
- Used for cultivation of fastidious organisms that
cannot grow on simple media and need highly
nutritive substances for growth.
-Used for culturing sterile body fluids such as blood or
CSF, where the finding of any organisms = infection
due to that organism. And also for primary
identification of microorganisms e.g. hemolysis on
blood
- e.g. blood agar, chocolate agar, Loeffler’s
serum .

10. 1
-
Blood agar
.
5-10%
sterile defibrinated sheep or human
blood + melted nutrient agar at 55°C
.
.
Red opaque solid medium
.
.
N. agar is sterilized in autoclave at 121°C for 30 min
.
Blood is added under complete aseptic condition at 45-
55 o
C
.
-
Supports the growth of most delicate organisms e.g
.
Streptococcus pyogenes
.
-
Identifying bacteria according to their haemolytic
action on the red cells
.

11. 2
-
Chocolate agar
.
Prepared as blood agar followed by raising
the temperature to l00 °C for 2 min. to rupture
red cells and release nutrients as X and V factors
.
.
Brown opaque solid medium
.
Sterilized as blood agar
.
.
Used for the isolation of Neisseria meningitides, Haemophilus
influenza and Streptococcus pneumonae

12. Blood Agar Medium
Plate
Chocolate Agar Medium
Plate & Slope

13. 3
-
Loeffler's serum
.
3
parts of sheep or horse serum
+
1
part glucose broth
.
Opaque whitish solid medium
.
.
Serum is sterilized by filtration & glucose
broth by Koch's steamer
.
.
The medium is solidified in hot air inspissator at
75°C for 2 hr for 2 successive days
.
Uses : Culture of Corynebacterium diphtheria

14. III. SELECTIVE MEDIA
. Solid media that contain substances (e.g. bile
salts or other chemicals, dyes, antibiotics) which
inhibit the growth of one organism to allow the growth
of another .
. Used when culturing a specimen from a site
having a normal microbial flora to prevent unwanted
contaminants overgrowing a pathogen.
. They include the following media:-

15. 1
-
Lowenstein Jensen medium
.
3
parts beaten eggs + 1 part water
+
malachite green
.
.
Green opaque solid medium
.
Sterilized in hot air inspissator
at 75 °C for 2hr for 2 successive days
.
.
Used for Isolation of Mycobacterium
tuberculosis

16. 2
-
MacLeod's tellurite blood agar
.
Blood agar + 0.02-0.04% K tellurite
.
.
Red opaque solid medium
.
Sterilized as blood agar
.
.
Used for isolation of Corynebacterium diphtheriae
from contaminated materials
.

17. 3
-
Modified Thayer-Martin agar
.
Chocolate agar + vancomycin + colistin +
nystatin
.
.
Brown opaque-solid medium
.
.
Sterilized as Chocolate agar
.
.
Used for Isolation of Neisseria from non
sterile specimens.

18. 4
-
Thiosulphate citrate bile sucrose agar
(
TCBS
)
.
Alkaline agar + sucrose + thiosulphate + citrate and
bromothymol blue indicator
.
.
Greenish transparent solid medium
.
.
Sterilized in autoclave at 121°C for 30 min
.
.
Used for isolation of Vibrio cholerae
.

19. TCBS medium

20. 5
-
Deoxycholate citrate agar (DCA
(
.
Agar + lactose + neutral red indicator
+
Na deoxycholate and citrate
.
.
Reddish semi transparent solid medium
.
.
Sterilized in autoclave at 121°C for 30 min
.
.
Used for isolation of Shigella and Salmonella
.

21. XLD Media
.
Agar + lactose + phenol red indicator + ferric
citrate + desoxycholate + xylose + lysine +
sucrose + yeast extract
.
.
Reddish semi transparent solid medium
.
.
Sterilized by boiling
.
.
Used for isolation of Shigella and Salmonella
.

22. IV. ENRICHMENT MEDIA
..
. Fluid media that contain substances which favour the
growth of wanted organisms on the expense of others.
. Usually used as a preliminary step for isolation of
pathogens before subculturing on solid selective
media. Examples are:
.
Selenite broth for isolation of Salmonella and
Shigella species from faeces
.
.
Tetrathionate broth for isolation of Salmonella
from faeces
.
.
Alkaline peptone water for isolation of Vibrio
cholerea
.

23. V. INDICATOR (DIFFERENTIAL)
MEDIA
. These are media to which dyes or other substances
(Indicators( are added to differentiate microorganisms.
. Indicators change colour when acid is produced
following fermentation of a specific carbohydrate e.g.
MacConkey's agar medium.

24. MacConkey's agar medium
. Peptone, agar, lactose, bile salt and neutral red
indicator.
. Reddish transparent solid medium
. Sterilized in autoclave 121°C for 30 min.
. Supports the growth of most Gram- negative bacilli,
especially the enterobacteriaceae, but inhibits the growth of
Gram positive organism and some fastidious Gram-
negative bacteria, such as Haemophilus and Neisseria.

25. MacConkey's agar
MacConkey's agar
medium
medium

26. VI. IDENTIFICATION MEDIA
These include media to which substrates or chemicals
are added to help identify bacteria isolated on primary
cultures. i.e. organisms identified must be first
isolated in pure culture.
Organisms are mainly identified by a change in the
colour of the medium and or the production of gas.
They include peptone water sugars, litmus milk, and
gelatin media.

27. 1
-
Peptone water sugar media
-
Peptone water + 1% tested sugar + 1%
Andrade's indicator
.
-
Durham tube is an inverted tube to visualize
gas bubbles produced from sugar fermentation
.
.
-
Yellowish transparent
-
Sterilized in Koch's steamer for 20 min on
three successive days (tyndallisation
(.
-
Used to test the biochemical activity of bacteria
on carbohydrates
.

28. 2
-
Litmus milk
-
Steamed fresh milk for 1 hour (cream is
removed) + litmus solution
.
-
Mauve
-
Sterilized in Koch's steamer for 20 min
on three successive days (tyndallisation
).
-
Used to test the saccharolytic activity of
bacteria
.
-
For identification of enterococci
(
bleaching
).

29. 3
-
Gelatin
-
10-15%
gelatin sheets dissolved in nutrient broth
.
-
The medium is solid below 24°C. Above this
temperature it melts into yellowish fluid
.
-
Sterilized in Koch's steamer for 20 min on three
successive days (tyndallisation
).
-
Used to test the proteolytic activity of
different organisms
.

30. VII. TRANSPORT MEDIA
- Semisolid media that contain ingredients to prevent
the overgrowth of commensals & ensure the survival
of aerobic and anaerobic pathogens when
specimens cannot be cultured immediately.
Examples:
1- Cary-Blair medium for preserving enteric
pathogens.
2- Amies transport medium for ensuring the
viability of gonococci
.
3
-
Thioglycollate broth and deep agar for
anaerobic organisms
.

31. CULTURE MEDIA FOR ANAEROBES
Media for anaerobes is the same as media for
aerobes except that:
1. They are richer in organic constituents .
2. Contain reducing agents (cysteine & haemin).
3. Contain a redox indicator .
The inoculated media are incubated in anaerobic
environment using anaerobic gas pack .

32. Robertson's cooked meat
Anaerobic enrichment media
-
5
gm cooked minced meat to which broth is added
.
(
Anaerobiosis is achieved through
reducing substances in the meat
e.g. haemin and glutathione
).
-
Sterilized in autoclave at 121°C
for 30 min
.

33. Anaerobic GasPak System
A method for the exclusion of oxygen from a
sealed jar used for incubation of anaerobic
cultures in a nonreducing medium .

34. Methods of isolation of pure culture
The streak-plate method to
obtain pure cultures

35. THANK YOU