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Bacterial Growth and

Enumeration
S. Y. B. Pharm Sem IV
2017-18
Ms. Heena Bhojwani
Bacterial Reproduction
• Can reproduce sexually and asexually
Methods for reproduction
1. Binary Fission
2. Budding
3. Fragmentation
4. Formation of conidiospores and
sporangiospores
Binary Fission
• Asexual process
• Results in division
of a cell into two
vegetative cells
• Also called as
transverse binary
fission
• Examples:
• Bacillus subtilis
• Streptococcus fecalis
Budding

• Small bud develops at one end of cell


• Bud enlarges and develops into new cell and gets
separated from parent
• Example: Hyphomicrobium species, Rhodopseudomonas acidophilia
Fragmentation
• Bacteria that produce filamentous growth
reproduce by fragmentation
• Filaments are fragmented into small bacilli or
cocci
• Example: Nocardia species
Formation of conidiospores and
sporangiospores
• Species of Streptomyces produce spores
• Develop crosswalls or septum at hyphal
tips
• Each spore gives rise to a new organism
GROWTH
• Growth: an orderly increase in all cellular
contents
• Increase in mass does not imply to growth
• Increase in mass: only size and weight
increase
• Growth occurs on cell division
• Increase in cell number reflects growth
Normal Growth Cycle (Growth
Curve) of Bacteria
• Bacterial growth: asexual reproduction or cell
division, of a bacterium into two daughter cells, in
a process called binary fission
• Providing no mutational event occurs, the
resulting daughter cells are genetically identical to
the original cell.
• “Local doubling" of the bacterial population occurs
• Both daughter cells from the division do not
necessarily survive.
• If the number surviving exceeds unity on average,
the bacterial population undergoes exponential
growth
Bacterial Growth Curve
Phases of Growth
• four different phases:
1. lag phase (no growth)
2. log phase or exponential phase (rapid
growth)
3. stationary phase (leveling off)
4. death phase (decline in growth)
Lag Phase
• Addition of inoculum to medium is not followed by
immediate growth
• Population: temporarily unchanged
• Bacteria adapt themselves to growth conditions
• Individual bacteria are maturing (grow in size beyond
normal dimension) and not yet able to divide
• Cells are not dormant
• Physiologically active, synthesize new protoplasm
• Synthesis of RNA, enzymes, coenzymes and other
molecules occurs
• Major metabolic activities occur and hence, no cell
division
• At the end of this phase, each organism divides
• But all may not complete this phase simultaneously
Log Phase
• Characterized by cell doubling
• If growth is not limited, doubling will continue at a
constant rate so both the number of cells and the
rate of population increase doubles with each
consecutive time period
• For this type of exponential growth, plotting the
natural logarithm of cell number against time
produces a straight line
• Populations are identical in composition of cells,
metabolic activity, and other physiological
characteristics
Log Phase
• Generation time or
doubling time varies
considerably among
different bacteria
• It can be calculated
from number of
generations “n” that
occur in a time
interval “t”
Log Phase
• During exponential growth, the rate of
growth is reciprocal of the generation time
• The slope of the straight line is the specific
growth rate of the organism
• It is given as
Log Phase
• Actual rate of this growth depends upon
the growth conditions, which affect the
frequency of cell division events and the
probability of both daughter cells
surviving
• Exponential growth cannot continue
indefinitely, however, because the medium
is soon depleted of nutrients and enriched
with wastes.
Stationary Phase
• Occurs due to a growth-limiting factor
such as the depletion of an essential
nutrient, and/or the formation of an
inhibitory/toxic product
• Stationary phase results from a situation
in which growth rate and death- rate are
equal
• Result is a smooth, horizontal linear part
of the curve during the stationary phase
The Phase of Decline or Death
• At death phase (decline phase), bacteria
die
• Caused mainly by lack of nutrients and
accumulation of toxic products such as acids
• Decrease in number of cells is exponential
• Inverse of the log phase
• Rate of death is different in different bacteria
• Example: some Gram –ve species die within
72 hours
Enumeration of Bacteria
• Determination of Cell Number
 Total count/ direct method
1. Direct microscopic count
2. Counting chamber method/ heamocytometer
method
 Viable count/ indirect method
1. Plate Count Technique
2. Membrane Filter Count
• Determination of Cell Mass
 Direct Method
1. Dry weight measurement
2. Measurement of cell nitrogen
 Indirect Method
1. Turbidimetric Method
• Determination of cell activity
 Indirect Method
1. Measurement of biochemical activity
Plate Count/ Pour Plate
method
• Most commonly used method for
enumeration of bacteria in a wide variety
of samples including milk, food, meat, soil
etc.
• Pour plate methods yield a count of only
the living cells
• Thus are a viable count
• Number of bacteria in a given sample is
usually too great to be counted directly
• There are two steps to the process:
1. Dilution of the sample so that various dilutions of the
sample may be inoculated onto plates and a count of the
colonies that grow made;
2. Plating of the dilutions so that each cell in the diluted
sample may then grow and form colonies that will in turn
become visible to the naked eye and can be counted

• Dilution is important:
colonies will have to be counted and with a concentrated sample
there may be too many colonies than can be accurately counted
• Plating is important:
count of only the living cells is required in this procedure (only
living cells will be able to multiply and form colonies)
Important considerations
• Only living cells are counted
• Keep in mind that if the organism normally forms
multiple cell arrangements, such as chains, the colony-
forming unit may consist of a chain of bacteria rather
than a single bacterium
• Some of the bacteria may be clumped together.
• Therefore, when doing the plate count technique, we
generally say we are determining the number of Colony-
Forming Units (CFUs) in that known dilution
• By extrapolation, this number can in turn be used to
calculate the number of CFUs in the original sample
• Bacterial counts by these methods are usually expressed
as colony forming units per milliliter (CFU/mL)
• Normally, the bacterial sample is diluted by factors of 10
and plated on agar.
• After incubation, the number of colonies on a dilution
plate showing between 30 and 300 colonies is
determined.
• A plate having 30-300 colonies is chosen because this
range is considered statistically significant
Procedure
1. Using sterile technique, transfer 1 mL sample to the first dilution blank.
Mix the bottle by inverting it 20 times. Label the bottle "10-1."
2. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the second bottle "10-2."
3. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the second bottle "10-2“.
4. Using a fresh pipette, transfer 1 mL from the first blank to the third blank.
Mix as before. Label the second bottle "10-3“.
5. Using a fresh pipette, transfer 1 mL from the first blank to the forth blank.
Mix as before. Label the second bottle "10-4“.
6. Using a fresh pipette, transfer 1 mL from the first blank to the second
blank. Mix as before. Label the fifth bottle "10-5“.
7. Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6, respectively.
8. Transfer liquid from the dilution blanks to the Petri dishes. Use a separate
pipette for each blank, not for each plate (i.e. if more than one plate uses
liquid from a single blank, a single pipette may be used for that blank).
9) One at a time, add a tube of molten nutrient agar
to each Petri dish. After adding the agar, gently
swirl the dishes in pattern for 30 seconds to mix
the bacteria with the agar.
10)After the agar has thoroughly solidified, incubate
the plates at 37°C for 24 to 48 hours.
11)Count the number of colonies on a plate that has
between 30 and 200 colonies.
12)Any plate which has more than 200 colonies is
designated as "too numerous to count" (TNTC).
13) Plates with fewer than 30 colonies do not have
enough individuals to be statistically acceptable
• Calculate the number of bacteria (CFU) per
milliliter or gram of sample by dividing the
number of colonies by the dilution factor
multiplied by the amount of specimen added to
agar plate.
• To compute the number of CFU/mL, use the
formula:
c = concentration, CFU/mL
n = number of colonies
d = dilution blank factor
s = volume transferred to plate.
Example for calculation
• You count 46 (n) colonies on your plate
• You put 1 ml of bacterial culture into 99 ml
of saline and plated 0.1 ml (s)
• Dilution 1/100 (d)
• CFU = 46/ 10-2 * 0.1
= 46*100*10
=46000 CFU/ml
Membrane Filter Technique
1. Collect the sample and make any necessary
dilutions.
2. Select the appropriate nutrient or culture
medium. Dispense the broth into a sterile
Petri dish, evenly saturating the absorbent pad.
3. Flame the forceps, and remove the
membrane from the sterile package.
4. Place the membrane filter into the funnel
assembly.
5. Flame the pouring lip of the sample container and pour the sample
into the funnel.
6. Turn on the vacuum and allow the sample to draw completely
through the filter.
7. Rinse funnel with sterile buffered water. Turn on vacuum and allow
the liquid to draw completely through the filter.
8. Flame the forceps and remove the membrane filter from the
funnel.
9. Place the membrane filter into the prepared Petri dish.
10. Incubate at the proper temperature and for the appropriate time
period.
11. Count the colonies under 10 - 15 X magnification.
12. Confirm the colonies and report the results.
Commonly used Filters
Advantages of Membrane Filter
Method
• Permits testing of large sample volumes.
• Reduces preparation time as compared to many traditional
methods.
• Allows isolation and enumeration of discrete colonies of
bacteria.
• Provides presence or absence information within 24 hours.
• Effective and acceptable technique. Used to monitor drinking
water in government laboratories.
• Useful for bacterial monitoring in the pharmaceutical,
cosmetics, electronics, and food and beverage
• industries.
• Allows for removal of bacteriostatic or cidal agents that would
not be removed in Pour Plate, Spread
• Plate, or MPN techniques.
Turbidimetric Measurement

• Quick and efficient method of estimating


the number/mass of bacteria in a liquid
medium is to measure the turbidity or
cloudiness of a culture and translate this
measurement into cell numbers/Cell mass
• Fast and is usually preferred when a large
number of cultures are to be counted
Turbidimetric Measurement

• Turbidity can be measured by an instrument


such as a colorimeter or spectrophotometer.
• These instruments contain a light source and
a light detector (photocell) separated by the
sample compartment.
• Turbid solutions such as cell cultures
interfere with light passage through the
sample, so that less light hits the photocell
than would if the cells were not there.
Turbidity Measurement
Turbidimetric Measurement

• Before turbidimetric measurements can be made, the


spectrophotometer must be adjusted to 100%
transmittance (0% absorbance).
• This is done using a sample of uninoculated medium.
• Percent transmittance of various dilutions of the
bacterial culture is then measured and the values
converted to optical density, based on the formula:
Absorbance (O.D.) = 2 - log % Transmittance.
• A wavelength of 420 nm is used when the solution is
clear, 540 nm when the solution is light yellow, and
600-625 nm is used for yellow to brown solutions.
Turbidimetric Measurement

• Much faster than the standard plate count but the


measurements must be correlated initially with cell number
• This is achieved by determining the turbidity of different
concentrations of a given species of microorganism in a
particular medium and then utilizing the standard plate count
to determine the number of viable organisms per milliliter of
sample.
• A standard curve can then be drawn (e.g., this lab protocol
section), in which a specific turbidity or optical density
reading is matched to a specific number of viable organisms.
• Subsequently, only turbidity needs to be measured. The
number of viable organisms may be read directly from the
standard curve, without necessitating time-consuming
standard counts.