DETERMINATON OF MICROBIAL POPULATION

Microbial Population
-is the number of colony-forming units (CFU), or viable microbial cells, present in a unit
volume or weight.
Importance
- Microbial counting is useful in the basic sciences and is used to determine the number of
bacteria present for physiological or biochemical studies - Routinely used in the areas of public
health. - Food or water microbiologists test food, milk or water for the numbers of microbial
pathogens to determine if these products are safe for human consumption.

SPREAD PLATE TECHNIQUE

A mixture of cells or of a cell culture is spread out on the Agar surface of a petri dish,
with the help of a flame-sterilized glass rod-made cell spreader and incubated over a defined
time period -usually between 12 and 48 hours. The surface-dispersed cells grow into distinctive
and visible cell clusters called colonies.
Advantages
It is useful for isolating aerobic microorganisms
Cultures are never exposed to 45C melted agar temperatures.
Simple to perform
No need for molten agar
No condensation
Disadvantages
It allows the growth of more microbes and presence of more colony forming units.
It doesnt allow the growth of obligate anaerobic microorganisms contamination of the
growth can occur.
Contamination of the growth can occur
Need good spread technique
Counting range
Plates must be dried just right
Spreaders

POUR PLATE TECHNIQUE

Small amount of inoculum from the broth culture was pipette in the plate. The original
sample is diluted several times to reduce the microbial population sufficiently to obtain separate
colonies when plating. The mixtures are poured immediately into sterile culture dishes. The
isolated cells grow into colonies and can be used to established pure culture
Advantages
For quantification of colonies in solid medium.
Allows the growth and quantification of microaerophiles because there is less oxygen
than on the agar surface
We can identify whether bacteria is an anaerobe, aerobe or facultative aerobe because the
anaerobe and facultative anaerobe will grow within the media.
Disadvantages
The temperature of the medium need to be carefully controlled: too hot will kill the
microorganisms, too cold will create clumps of congealed agar, which can sometimes be
mistaken for colonies
There is problem of making sure the colonies spread evenly in the medium
The colonies can overlap in the agar which can be difficult in picking without mixing.
The aerobes also trapped inside the agar, low or no oxygen penetrate or diffuse into the
agar, causing aerobes fail to survive. Therefore, pour plate method is considered as
selective method

TURBIDIMETRIC METHOD

McFarland Standards has a chemical composition of 1% barium chloride and 1% sulfuric

acid. These two chemicals when added, fine precipitate will be produce. Barium sulfate will
depend on the amount of reactant to produce specific optical density. This optical density is
comparable to turbidity of cell suspension of culture. This standard is one of the most widely
used methods of inoculum standardization.
Advantage
There is no incubation time or equipment needed to estimate bacterial numbers.
It is convenient to use.

Disadvantage
Interpreting the turbidity in subjective manner may give inaccurate result.
Valid only for gram negative bacteria.

MILES AND MISRA METHOD

Advantages
This technique is faster than other methods.
 With this technique, less bacterial contamination occurs of the working surface.
This technique is easy to process as compared to other techniques for determining colony
forming units.
Disadvantages
This technique required high skilled microbiologist to perform serial dilution and also the
one who are expert in aseptic techniques.
The rate of absorption of drops on to the surface of agar depends upon the environmental
conditions like temperature and humidity.