MEASUREMENT OF BACTERIAL

GROWTH (Bacterial populations)

A. Methods for Measurement of Cell Numbers
• -- Knowledge of numbers of bacteria present in a water/soil
samples is more important to the engineer/scientist than
knowing the types bacteria present.
• -- He needs to know the number of a certain bacterium or group
present as well as the overall number of all types of bacteria.
Measuring techniques involve:
• 1) direct counts, visually or instrumentally,
• 2) indirect viable cell counts
1) Direct microscopic counts:
are possible using special slides known as counting chambers.
• -- However, dead cells cannot be distinguished from living ones
during microscopic count.
2) Indirect viable cell counts/ plate or colony counts:
Counts on solid media
• -- this is done by making counts on solid media
Viable cell:
- is defined as the cell/one that is able to divide & form offspring
when cultured under appropriate conditions.
- Viable cell count involves plating out (spreading) a sample of a
culture on a culture medium e.g. nutrient agar surface.
- The sample or cell suspension can be diluted (see serial dilution
technique) in a nontoxic diluent (e.g. water or saline) before
plating.
- If plated on a suitable medium, each viable unit (cell) grows and
forms a colony.
• Each colony known as a colony forming unit (cfu) can be
counted & the number of cfu's is related to the viable number of
cells in the sample.
Advantages of the technique are:
i) high sensitivity (a single cell can be detected

• Disadvantages:
i) only living cells develop colonies that are counted;
ii) clumps or chains of cells develop into a single colony;
iii) colonies develop only from those organisms for which the
cultural conditions are suitable for growth.
 There are 2 main ways of
performing viable/plate counts:
a) The spread plate method
b) The pour plate method

Figure 1. Bacterial colonies growing

on a plate of nutrient agar.
a) The spread plate method
• -- a volume of diluted sample or cell suspension no larger than
0.1 ml is spread over solidified agar surface, using a sterile
glass spreader.
• The plate is incubated until the colonies appear & the number of
colonies is counted

b) The pour plate method

• -- a volume of diluted sample (of known volume) is mixed with
melted agar in plate before it cools & hardens.
• Upon incubations, surface & sub surface colonies are formed.

Note: It is important that in both pour plate & spread plate, the
colony count be done on plates that have between 30 – 300
colonies.
ENUMERATION OF BACTERIA
• Standard plate count/ colony forming unit (CFU)is one of the most
common methods for determining bacterial numbers in a sample.
• As a sample is diluted in a series of dilution blanks as shown
below. Aliquots of the dilutions are then plated onto media and
the numbers of colonies are counted after incubation for 24-48
hours.
• It is assumed that the bacterial cells are diluted to an end point
where a single cell divides giving rise to a visible colony on a plate.
• The number of bacteria in the original sample is determined by
multiplying the number of colonies by the dilution factor.
Example on calculating the CFUs
• First step: work out on the total dilution of the sample. Thus, f irst 5mL is added to 45mL; which is
1/10 dilution.

• I.e. 5ml of the sample / (5mL + 45mL) = 5/50= 1/10

• Now, the initial dilution is 1 to 10 dilutions.

• Second: 1ml in 99ml which is 1/100 dilutions.

• I.e. 1mL/ (1mL +99mL) = 1/100 = 1/100

• Third: 1ml is again added to 99ml; which is 1/100 dilutions.

• I.e. 1ml/ (1mL + 99mL) = 1/100 = 1/100

• Note; less than 1ml is put on the plate is also kept into consideration. In this problem 0.1ml is equal
to 1/10.
Now calculating total dilution,
• 1/10 x 1/100x 1/100 x 1/10 = 1/10 = 10
6 -6

Calculating CFU from dilution plating results: how does a count

on plates get converted to CFUs per gram or mL of sample?
• The next step is to work out with the dilution factor.
Note; the dilution factor is the reciprocal of total dilution.
I.e. 1/10 = 1÷1/10
-6 6

= (1 x10 )/1 6

= 106

Finally multiply the total dilution factor by the average number of

colonies in a plate/Volume plated.
I.e. CFUs/ ml =X colonies x 10 / mL
6

=X x 10 x 106 1

X x 10 CFUs/ mL
7
Counts in Liquid Media (Broth)
• This method is not as accurate as counts on solid media
- They are employed whenever the observation of gas production
is an
integral part of the test.
• Usually, selective media are employed for bacterial counts.
• The most commonly used technique for enumeration of
bacteria (mainly coliforms) in liquid culture (broth) is known as
the Multiple tube fermentation or the most probable number
(MPN).
- The resulting pattern of positive & negative results in dilution
series is used to obtain a statistical estimate called MPN of the
cells per stated volume of sample.
• The MPN is based on the assumption that each positive tube
received at least one viable cell.
- The MPN can be simply read/obtained directly from the
probability tables

• Most probability tables:

--The MPN tables show the MPN corresponding to various
combinations of positive results, including the 95% confidence
limits(CLs).
-- 95% CLs indicate the lower and the upper limits between which
the real density of coliform organism is expected to fall with the
probability of 95%.
• In multiple tube procedure, usually five tubes are employed.
-- When more than three dilutions are employed in a decimal
series of dilutions, the results from only three of these are
significant.
--The three dilutions to be used in determining the MPN, using the
five tubes of each dilution are selected as follows:
• “[The highest dilution which gives positive results in all of the
five culture tubes tested (no lower dilution giving any negative
results) & the two next succeeding higher dilutions should be
chosen]”
• In the absence of MPN tables or for combinations that do not
appear in the table and for other combinations of tubes or
dilutions, MPN can be calculated Thomas’ simple formula:
Membrane Filtration (MF)
• Membrane filters are thin porous sheets of cellulose esters
whose porosity can be controlled during manufacture
- All object that are bigger than the pore size are retained on the
filter.
- Most bacteria are retained on a filter of pore size 0.45 µm
diameter & all bacteria on a filter of 0.22 µm pore diameter.
A known volume of the sample is filtered through a sterile
filtration apparatus & the membrane is aseptically transferred
either to a small Petri dish with a tightly fitting lid containing a
sterile absorbent pad saturated with a suitable medium or to a
Petri dish containing an agar medium.
- Visible colonies formed on a thin layer of nutrient on the
membrane filters are counted.
Advantages of MF:
a) Enables large volumes to be examined.
b) Shorter incubation times/period
c) More accurate colony counts than MPN

Disadvantage:
a) Turbid waters clog the membrane & hence interfere the
filtration process
b) Membranes are expensive hence should be used only if the
counts expected is less than 30 cells per mL
CULTURE MEDIA
What is culture medium?
• Are food material or substances required for growing
microorganisms in vitro (outside the body).
Uses of culture medium
i. To identify the cause of infection from the clinical sample, so
that proper treatment can be given
ii. To study the characteristics or properties of microorganisms
iii. To prepare biological products like vaccine, antigens, and
toxoids.
iv. To isolate and identify bacteria, reveal their metabolic
properties, and allow long-term storage of pure cultures.
Composition of culture media
i. Water
ii. Energy source
iii. Nitrogen source
iv. Mineral salts
v. Special growth factors.

Types of culture media

Classification based on physical state
i. Solid medium
ii. Semi- solid medium
iii. Liquid medium
Classification based on the ingredients
• i. Simple medium
• ii. Complex medium
• iii. Synthetic or defined medium
• iv. Special media

Classification based on physical state

Solid medium
Agar is the most commonly used solidifying agent
Semi solid media
Such media are soft and are useful in demonstrating bacterial motility
and separating motile from non-motile strains.
Liquid media
Are sometimes referred as broth
Bacteria grow uniformly producing general turbidity. E.g. Nutrient
broth.
Classification based on the ingredients
Simple media
E.g.: Nutrient broth, Nutrient agar
Complex media
Such as blood agar, it has ingredients that exact components are
difficult to estimate.
Synthetic or defined media
Specially prepared media from pure chemical substances for
research purpose and composition of every component is well
known.
• E.g.: Peptone water ( 1% peptone + 0.5% Nacl in water)
Special media
• Enriched media
• Selective media
• Differential media
• Transport media
• Anaerobic media

Enriched media
Used to grow bacteria that are exacting in their nutritional needs.
E.g. Blood agar, chocolate agar.
Selective media
• The inhibitory substance is added to a solid media to inhibit
commensal or contaminating bacteria such as: Antibiotics, Dyes,
Chemicals and Alteration of pH
• Examples: Thayer Martin medium, Eosin methylene blue,
Campylobacter agar, Lowenstein- Jenson medium.
Differential media
• Are designed in such a way that different bacteria can be recognized
on the basis of their colony color.
Examples: MacConkey agar
Transport media
• Media used for transporting the samples. E.g. Stuart’s medium,
buffered glycerol saline.
Anaerobic media
• These media are used to grow anaerobic organisms.
• E.g. Robertsons cooked meat medium, Thioglycolate broth medium.
B. Methods for Measurement of Cell Mass

• Methods for measurement of the cell mass involve both direct

and indirect techniques:

1. Direct physical measurement of dry weight, wet weight, or

volume of cells after centrifugation.

2. Direct chemical measurement of some chemical component

of the cells such as total N, total protein, or total DNA content.

3. Indirect measurement of chemical activity such as rate of O2

production or consumption, CO2 production
or consumption, etc.
• 4. Turbidity measurements employ a variety of instruments to
determine the amount of light scattered by a suspension of
cells.
• - Particulate objects such as bacteria scatter light in proportion
to their numbers.
• - The turbidity or optical density of a suspension of cells is
directly related to cell mass or cell number, after construction
and calibration of a standard curve.
• The method is simple and nondestructive, but the sensitivity is
limited to about 107 cells per ml for most
 PURE CULTURE (Growth of individual organisms)
• A pure culture is defined as the progeny (clone) of a single cell or a culture
that has arisen from a single cell.
- The isolation of pure cultures (i.e. of a given organism) is carried out on or in
solid media by either:
(a) agar streaking:
• - This procedure involves preparation of Petri dishes containing a suitable
culture medium solidified with agar.
- a sterile loop is placed in a mixed culture containing the organism of interest
& then lightly streaked across the surface of the agar plate
- The streaked plate is incubated so that the organisms will multiply &
produce colonies.
- One of the well isolated colonies is then restreaked on a fresh agar plate &
incubated.
- If all of the colonies obtained are of similar size, shape, color & texture, it is
presumed that they are all alike & that a pure culture has been obtained.
b) Pour plates.
- A diluted inoculum/sample is mixed with the melted agar in the
plates before it cools & hardens. When the inoculated poured
plates are incubated, isolated colonies should be obtained from
which pure cultures are prepared.
c) Membrane filtration:
- A membrane filters are used as the solid support instead of agar.
- A dilution of inoculum can be passed through the filter & the
filter is then placed on an appropriate culture medium for
incubation.
- Isolated colonies developing on the filter can then be picked to
prepare pure cultures.
 Gram stain
• Gram stain (Gram staining or Gram's method), is a method of staining used to
classify bacterial species into two large groups:
i. gram-positive bacteria
ii. gram-negative bacteria.
It may also be used to diagnose a fungal infection.
• Gram staining differentiates bacteria by the chemical and physical properties of
their cell walls.
• Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains
the primary stain, crystal violet.
• Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet
to wash out on addition of ethanol. They are stained pink or red by the counterstain,
commonly safranin or fuchsine. Lugol's iodine solution is always added after
addition of crystal violet to strengthen the bonds of the stain with the cell
membrane.
• Gram staining is almost always the first step in the identification of a bacterial group.