Microbiological culture & colony count

Microbial growth

Culture media, Oxygen,

Chemical requirements

Temperature, PH,
Isotonic solution
Binary Fission
Bacterial colony

A visible mass of
microorganisms which
originating from a
single mother cell.
 Microorganisms culture
Two basic culture techniques used:

 Liquid culture: Bacteria, algae, and some fungi can be

reared in culture tubes (test tubes) in a liquid medium.

 Culture Plates: Liquid medium is solidified using agar

(agarose) and poured as a thin layer in the bottom of a

culture plate (also sometimes called petri plate)

 Sterile technique

Washing lab benches with a surface disinfectant.The most

commonly used disinfectants for lab use are:

 10% bleach (recommended by the CDC)

 70% ethanol
 Sterile technique

 Sterilize wire loop/Inoculating loop

 Neck of the test tubes and replacing cap

 Sterile pipettes must not be touched to clothing,

the surface of the working area

 Isolation of pure culture

Technique used to isolate pure culture

 Streak plate

 Spread plate

 Pour plate
 Purpose of streaking 

 To produce isolated colonies of an organism (mostly

bacteria) on an agar plate

 Purification of isolate particular strain of bacteria from

contaminants.

 To identify the organism

 Considering technique during inoculum

 Use only a small amount of inoculums

 Streak lightly so that not gouge the agar.

 Flame the loop after streaking each quadrant.

 Make sure surface of the plate is free of droplets of

condensed moisture
Quadrant streaking for isolation pure culture
 Streak plate procedure

 Sterilize the inoculating loop in bunsen burner

 Pick isolated colony from the agar plate culture

 Spread it over the first quadrant (approximately 1/4 of the plate) 

 Flame the loop again and allow it to cool

 Streaking on agar plate by inoculating loop in 2nd, 3rd & 4th

quadrant
Streak on MAC & Nutrient agar
 Spread plate method
• Generally 0.1ml of the diluted sample is pipetted
onto the surface of a solidified agar medium

• Spread with a sterilized, bent, glass rod.

 Pour plate techniques
 Counting the number of colony-forming bacteria present
in a liquid specimen

 Generally 1 ml from a broth/sample is placed in the

center of sterile petri dish using a sterile pipette.

 Molten cooled agar (approx.15mL) is then poured into

the Petri dish

 After the solidification of the agar, the plate is inverted

and incubated at 37°C for 24-48 hours.
 Pour plate
 The pour plate method of counting bacteria is more
precise than the streak plate method

 But, on the average, it will give a lower count as heat

sensitive microorganisms may die when they come
contact with hot, molten agar medium.

 The pour plate technique used to determine the number

of microbes/ml in a specimen

 Used to assay bacterial contamination of feed stuffs.

Methods of isolating pure culture
 Estimating microbial population
 Plate are incubated at 37°C for 24 hours

 Examine the colonies grown in the plate carefully

 All colonies should have the same general appearance

 If using a commercial plate counter, touch each colony

on the plate with the pen, and the cumulative number of
colonies will appear on the display.
 Pour plate method
Materials and Equipments
 Test sample
 Plate Count Agar (PCA) or Nutrient Agar
 Hot water bath 45°C
 Sterile Petri dishes
 Flame
 Colony counter with magnifying glass
 Sterile capped
 Pipettes of various sizes (e.g. 1.0 and 2.0 mL)
 Pour plate method
Procedure of Pour plate technique:
 Prepare the dilution of the test sample expected to
contain between 30-300 CFU/ml
 Inoculate labeled empty petri dish with specified ml

(0.1 or 1.0 mL) of diluted specimen

 Pouring the molten agar and incubation

Collect one bottle of sterile molten agar (15 mL) from the water bath (45°C)

Hold the bottle in the right hand; remove the cap with the little finger of the

left hand.

Flame the neck of the bottle.

Lift the  lid  of  the  Petri  dish  slightly  with the  left  hand  and  pour  the 

sterile  molten agar  into  the  Petri  dish  and  replace  the the lid.


Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.
 Pouring the molten agar and incubation
 Flame the neck of the bottle and replace the cap

 Gently rotate the dish to mix the culture and the medium

thoroughly and to ensure that the medium covers the plate

evenly.

 Allow the agar to completely gel without disturbing it, it

will take approximately 10 minutes.

 Estimating microbial population size

 After 24-48 hours, count all the colonies. A

magnifying colony counter can aid in counting

small embedded colonies.
 Calculate CFU/ml using the formula:
CFU/mL=CFU *dilution factor /diluted
sample(ml)
Pour plate: E.coli on Mackonkey
 Disadvantages of Pour plate method

 Preparation for pour plate method is time consuming.

 Loss of viability of heat-sensitive organisms
 Embedded colonies are much smaller than those which
happen to be on the surface.
 Reduced growth rate of obligate aerobes in the depth of
the agar.
 Serial Dilution of Samples

 Serial dilution techniques used in the estimation

of microbial population sizes.

 Serial dilution A serial dilution is a series of

sequential dilutions used to reduce a Bacterial

cell to a more usable concentration.

 A Ten fold dilution of a sample
 Successive dilution of a sample.
 Process of reducing concentration of a
solute/sample in solution.
 Also known as “viable cell count”

1 ml 1 ml
1 ml 1 ml

Stock solution
9 ml diluent
25gm+225 ml 9 ml diluent 9 ml diluent
9 ml diluent
diluent

1:10 1:100 1:1000 1:10,000 1:1,00000

 Serial dilution & estimating microbial population

Stock solution
9 ml 9 ml
25gm+225 ml 9 ml 9 ml
diluent diluent
diluent diluent diluent

 Calulation: No of colonies on plate x Reciprocal of Dilution Factor

on plate=No. of Bacteria/ml= eg. 30 x 102 =3000 CFU per ml
 Serial dilution & estimating microbial population

Serial dilution- Serial dilution- Serial dilution- Serial dilution-

01 02 03 04

Dilution=Stock
volume/Total
volume 1/1+9=1/10= 1/1+9=1/10= 1/1+9=1/10= 1/1+9=1/10=
0.1=1x10-1 0.1=1x10-1 0.1=1x10-1 1x0.1=10-1

Final dilution 1x10-1 1x10-2 1x10-3 1x10-4

Dilution factor 10 100 1000 10000

 Calculating the number of bacteria per ml of
serially diluted bacteria

• To calculate the number of bacteria per mL of diluted sample :

 Number of CFU
Volume plated (mL) x total dilution used

• For example, if 1x 10-4 dilution plate, you plated 1mL of the

diluted cell suspension and counted 200 bacteria, then the
calculation would be:
 
• 200/1 mL x 10-4 or 200/10-4 or 2.0 x 106 bacteria per mL.
 
Colony counter machine
 Possible errors in plate counting

 Suitability of culture medium

 Length of incubation and incubations conditions.

 Some tiny colonies may be missed during

counting
 The incubations condition
(Medium,Temperature,Time)
 Key dilutions must be prepared
 References
 Basic Practical Microbiology A Manual by Society for

General Microbiology (SGM)

 https://microbeonline.com/pour-plate-method-principle-

procedure-uses-dis-advantages