BACTERIAL GROWTH MEASUREMENT

Growth means an increase in number, mass, size of cells. Bacterial growth usually refers to
reproduction.

Increase in population rather than enlargement of cells reproduction in bacteria occurs mainly
through binary fission or transverse fission. The time taken by a population of bacterial cells to
double its population is called doubling time or generation time. The length of generation time is a
measure of the growth rate of an organism. The average time in bacteria is 30 – 60 mins under
optimum conditions.

Staphylococcus aereus and Salmonella enteriditis are example of pathogens having relatively short
doubling time (20 mins).Mycobacterium leprae (30 days) has the largest generation time.

Determination of bacterial growth requires inoculation of the culters under optimum temperature
and gaseous conditions.

The bacteria will reproduce rapidly .Under this condition of microbial growth can be measured by
the fallowing method.

1) Turbidometric method

2) Cell count by haemocytometer

3) Standard plate count method.

1) TUBIDOMETRIC METHOD

Aim: To measure the growth of bacteria by Turbidometric method.

Priciple: Turbidometric method is an indirect method of determinig the microbial mass by light
absorption optical density is directly proportional to all concentration .AS THE POPULATION On
and turbidity increases. The absorbance is increases light are passes through the medium whose
intensity can be measured by spectrophotometer.

The bacterial growth curve shows 4 distinct phases of growth.

Lag phase-During this phase the number of cells remains the same but considerable metabolic
activity occurs.

Log phase-In this stage bacteria begins dividing and therefore cell number increases exponentially,
it is called exponential phase.

Stationary phase-In this phase the population remains constant for a time since the reproduction
rate is balanced by an equivalent death rate.

Death phase-The bacteria die at a faster rate than the faster than the formation of new cells,
therefore the number of viable cells decreases exponentially

REQUIREMENTS
10 -12 broth culters of E.coli maintained at log phase by immersion in an ice bath.

Cuvetts and spectrophotometer /colorimeter.

Pipette, conical flask .

Nutient broth

PROCEDURE;

1. Aseptically transfer nutrient broth into 5 conical flasks (100 ml flaks 0 and sterilise by
autoclaving at 121 c for 15 mins at 151 lbs.

2. About 0.5 ml of 12-24 hours broth cultures of E.coli was added to all the conical flasks after
bringing it to room temperature

3. Contents were mixed well for 10 mins in shaker and incubate all conical flask in incubation at 37
C for a interval of 30 mins,1 hour,2 ,3 ,4,5,6,7,24,30,48 hours .

4. 0 min the media, the media contains only 0.5 ml of media inoculum.The spectrophotometer
reading was taken against uninoculated media as blank.

5. After respective hours ,the conical flasks with added inoculum was measurement 620nm.

6.The graph of the time v/s absorbance at 620nm was plotted.

OBSERVATION:

CELL COUNT METHOD

AIM: To measure the growth of bacteria by cell count method.

Principle: Haemocytometer is instrument used to mainly count the number of cells and estimate
their concentration. it consist of a their glass slide having depression at the centre on both sides
of which there is one groove and a cover slip

In the centre of depress portion there is a grid of definite area which is divided into 25 squares
with 1mm slide. The centre square is further divided into 25 small squares ,each square having 0.2
mm each of the squares are divided again into 16 squares each having 0.5 mm slide. In this small
square, counting is done .since the distance between the counting and the central depression is
just 0.1 mm.it is easy to enumerate the number of microorganisms in a known volume of
suspension introduced.

REQUIREMENTS:

Nutrient broth media, bacterial culters , flask etc...

PROCEDURE:
1. Slide was cleaned by alcohol to remove grease .10-4 diluted sample of bacterial suspension of
produced by means of a pippeting syringe to prevent flow of extra suspension.

2. coverslip was placed over the suspension and pressed with thumb to ensure a uniform depth
over the counting area .

3. The slide was placed on the microscopic stage and allowed the cell to settle and the light was
focused.

4. The number of cell was counted in at least in 5 small squared separately. The average in each
square was calculated.

5. The slide should not be allowed for a long period to be counted because this will affect counting
due to evaporation from the suspension.

6. The number of cells per ml of the suspension was calculated.

RESULT:

PLATE COUNT METHOD

AIM: To measure the growth of bacteria by plate count method .

Principle: It is the one of the most routinely used produce because of the enumeration of viable
cells by this method.this method is based on the principle that when material containing bacteria
is cultured,every viable bacteria developed into a viable colony on a nutrient agar medium
.therefore the number of colonies are same as the number of oraganism (individual cells) in the
sample dilutions are usually made in the multiple of 10 single dilutions is calculated as
Dilution=volume of sample by total volume of sample and dilution.

Serial dilution are preapared by transferring a known volume of dilution to second dilution,blank
and so on.once diluted the specified volume ,of the dilution sample from various dilutions is
added to serially petriplates to which molten and cooled suitable agar medium is added .The
colonies are counted on acidic colony counter.The number of organisms developed on the plate on
incubation priod of 34-48 hours is obtained by multiplying the number of colony obtained per
plate by the dilution factor which is reciprocal of the dilution.

Number of cells /ml=no of colonies by amount plated *dilution.

PROCDURE :

1.Dilutions are labelled as 10-1,10-2,10-3--------10-7.

2.Initial dilution is prepared by adding 1 ml of sample into 9 ml dilution.

3.10-1 labelled is diluting the original sample 10 times.

4.The contents are mixed to obtain uniform distribution of organism .

5.Transfer 1 ml of suspension from the 1st dilution to the 2 nd and successively upto 10-7 .
6.From the appropriate dilution 1 ml of suspension was transferred to sterile petriplates and 15 ml
of nutrient agar medium was added to each petriplate containing sample .

7.The contents were mixed well by rotating gently and the plates are allowed to solidify

8.The plates are the incubated in an inverted position for 24-48 hours at 37 degree C .

RESULT: