Lab:5
Microbial growth in the laboratory
Culture media
Media: which is used for cultivation of bacteria in order to diagnose the
bacteria by the culture characteristics.
The basic requirement of culture media are:1- Energy source (carbon source).
2- Nitrogen source .
3- Salts .
4- Minerals.
5- Water.
6- Adjust PH (7.2 to 7.6) .
7- Adequate oxidation – reduction potential .
In general we can say that culture media are for:
1- Isolated of pure culture .
2- Collection of sample .
3- Growth of microorganism .
Media are solidified by the addition of solidifying agents such as agar.
Agar – agar : A complex carbohydrate obtain from Alage used as
solidification agent for media. Agar dissolved in aqueous solution,
melts at 100 °C, solidifies at 42°C but will remain liquid at 60 °C .
agar has no source of nutrients to the bacteria .
Table : Differentiation between agar and Gelatin .
Agar
Gelatin
1. Gelatin found in 15% in the
1. Agar found in 1.5% in the
medium so it is expensive
medium so it is cheap
2. Agar remain unmelted at all
2. gelatin melt at 25°C
incubation tempreture
3. Agar not decomposes by
3. gelatin decomposes by bacteria
bacteria
Preparation of culture media:
1. Dissolving powder media in distilled water and mixing of heater to
boiling.
2. Sterilization of media in auto clave at 121°C and pressure 15 pound
for 15-20 min.
3. Cooling the media and adding the media to petridishes or vials
,wait
until
solidify,
plates
are
kept
inverted
The culture media divided according to physical state to:1- Broth media : used as transport media and for cultivation of
bacteria. Contain zero% of agar. Organisms grown in broth
cultures are apparent through the turbidity that the large numbers
of cells produce in the broth.
2- Semi solid media : used for study the motility of bacteria. Contain
0.2 – 0.5 % agar. On agar, a solid medium, the bacterial cells form
masses called colonies after about 18 – 24 hours of growth
3- Solid media : used to study the all culture characteristics of colony
and contain 1-2% agar to solidify the media.
Classification media according to the composition :
1. Complex media : which is the exact chemical composition is
unknown example : plant, animal tissue, egg.
2. Synthetic media : which is the exact chemical composition of
medium is known : example: MacCo key agar, Nutrient agar.
3. Semi synthetic media : in these media the basic media (synthetic
media) added a complex substance example : blood, serum,
peptone.
Main Types of Media
1-basic media :
examples
nutrient
agar
nutrient
broth
They are usually used for preparation of enriched media
To sub-culture pathogens from differential or selective media
To do susceptibility testing which used for all groups of bacteria
 Nutrient broth (Beef extract, peptone, D.W)
 N. agar (N.broth + agar 1-2%)
 Semi solid (N.broth + agar 0.2-0.5%
2-Differential media : The addation of certain reagent or chemical
to the media may help in differentiating of certain types of bacteria
from others.
Ex: MacConkey agar : To differentiate between lactose
fermentation and Non lactose fermentation bacteria.
Fermentation of bacteria will below the PH of media by producing
acids, the PH indicator Neutral red will recognize the colony to :
Lactose fermentation colony
pink color
Ex: E.coli , klebisella spp.
Non lactose fermentation colony
Ex: Salmonella spp.
pale or colorless
Proteus spp.
3-Selective media : in this media allow the isolation of specific group
of bacteria and contain chemical substances that inhibit the growth of
one type of bacteria.
For example : crystal violet or bile salt will prevent growth of gram
positive bacteria without affecting the growth of gram negative
bacteria.
Example of selective media are :A- MacConkey agar : selective media because contain bile salt
which inhibit G+ and allow the growth of G- bacteria .
B- Salmonella – shigella agar
ssagar
Selective medium inhibition the growth of all bacteria and allow to
growth of salmonella and shigella
Selective : high bile salt
Sodium citrate
inhibition G+
G- (coli form)
And we can differentiate between salmonella and shigella on
ssagar
Neutral red (indicator)
Salmonella
Shigella
brown colony with black center
pale or colorless
C-Mannitol Salt agar
contains 7.5% Nacl which inhibition the growth of all bacteria
except staphylococci Spp and we can differentiate between Staph
spp on this media:
Staph aureus
manitol
Yellow color by fermentation of
4-Enrichment media
Which is used for fastidious bacteria and used by adding blood as a
rich substance to the nutrient agar
Example:
A- Blood agar
Prepare by adding 10% of sterile blood to sterile Nutrient agar
at 50°C used human blood or sheep blood.
Blood agar Enrichment and differential because it different
between B – haemolysis Str. Pyogens
And α – haemolysis Str. Viridians
B- Chocolate agar
Prepare by :
A. Adding 10% of sterile blood to the heated Nutrient agar
(more than 50°C) and the RbCs will lysis and V and X
factors are relased to the media.
B. By putting the blood agar in oven or in incubator at 55 °C or
more for 1-2 hours.
5-Special media
Used for one type of bacteria
For example: Lowenstein – Jensen medium used for the cultivation
of Mycobacterium tuberculosis
This contain Malachite green which inhibition all groups of
bacteria except Mycobacterium sp. and contain
inhibit M.bovis and allow to growth M.
glycerol
tuberculosis
Classification according to O2 requirements:
1. Aerobic media
All types of media like nutrient agar , N.broth, MacConkey agar,
blood agar, chocolate agar ……. Etc)
Which incubate in incubator in aerobic condition
2. Anaerobic media
A. These media are used to grow anaerobic organisms.
Ex:, Thioglycolate medium.
B. : There are special technique for cultivation of anaerobic bacteria:
1. Candle jar : a burning candle should use up all the O2 before it
gets extinguished and provides concentration of carbon
dioxide.
2. Macintosh and field jar : inoculated culture plates are placed
inside jar and using Gas pak by adding 10ml of water
2H2O + 2NaHCo3
2H2Co3 + 2NaOH
unstable
2H2 + 3Co2