Transcript lab7

PHT 226
Lab number 7
Total and viable
count of bacteria
IMPORTANCE OF BACTERIAL COUNT
VARIOUS METHODS ARE USED IN
MICROBIOLOGY TO MEASURE THE
NUMBERS OF MICROORGANISMS PER
UNIT VOLUME OF A GIVEN SAMPLE
This measurement is needed for:1. Standardization of inocula in
microbiological assay
[e.g. evaluation of antimicrobial
agents, assay of vitamins]
1. Industrial fermentation.
2. Evaluation of sterilization
technique.
HOW TO DETERMINE BACTERIAL
GROWTH?
1. DIRECT MICROSCOPIC COUNT:
USED TO DETERMINE THE NUMBER OF
BOTH DEAD AND LIVING BACTERIAL
CELLS (HAEMOCYTOMETER).
2. Turbidimetric determination
(viable & dead)
INCREASED TURBIDITY IN A CULTURE IS ANOTHER
INDEX OF BACTERIAL GROWTH AND CELL
NUMBERS.
THE INCREASE IN THE NUMBER OF CELLS DURING
GROWTH INCREASES THE TURBIDITY.
TURBIDIMETRIC DETERMINATION IS DONE USING A
SPECTROPHOTOMETER.
SPECTROPHOTOMETER

Measure growth rates with a
spectrophotometer

Direct relationship between
cell number and
absorbance, Otherwise
known as Optical Density
 More bacteria= higher
absorbance.
 less light reaches sensor
 Cells scatter light, not
absorb light
•Measure living and dead cells
•Gives immediate assessment
of the number of cells in a
population.
How to USE THE SPECTROPHOTOMETER
Bacterial suspensions are measured at wave length equals
600 nm .
 A clear solution will allow almost all of the light through
(BLANK).
 Light entering a cloudy solution will be absorbed.
 The amount of absorbance obtained measures what fraction
of light passes through a given solution and compared to that
absorbed by a clear solution.
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 The
amount of cells in
the solution is
directly proportional
to the absorbance
reading (linear
relationship)

A graph of
absorbance vs.
concentration will
give a straight line.
3. Dry weight and nitrogen
content determinations:
 In
this method, the bacterial cells
are collected by centrifugation,
then dried in an oven overnight at
85℃.
 The dry weight of bacterial mass
will be proportional to their
number.
 Also the nitrogen content of the dry
sample can be determined by micro
kjeldahl method.
MICRO KJELDAHL
METHOD
 Bacterial
cells are heated with conc
H2SO4
 Ammonia is liberated.
 Ammonia is distilled & captured in
boric acid solution.
 Titrate with 0.01N HCL
 Using methyl red- bromo cresol
green as indicator.
4. Measurement of microbial
activity:
Many microbial activity measured
quantitatively and used as a measure
of microbial growth.
e.g; the growth of acid forming
bacteria may be measured by simple
titration of the culture using
standard alkali.
5. Viable count of bacteria:
in this method only viable cells which
are capable of reproduction are
counted.
Principle:
Based on the fact that if the viable
cells are allowed to grow apart from
each other on a solid medium, each
cell develops into one visible colony.
The number of colonies obtained is
equal to the number of viable cells.
VIABLE COUNT OF BACTERIA

There are different procedures used to
determine viable bacterial count:-
a. Pour plate method.
b. Spread plate method.
c. Surface viable count.
A. POUR PLATE METHOD
In this method different dilutions are done from
the bacterial suspension.
 1 ml of each dilution is then poured on a sterile
empty Petri dish.
 15 ml of melted nutrient agar whose temperature
is about 45oC is poured in each plate with
swirling.
 The plates are left to dry and then incubated at
37oC for 48 hours.
 The
plates containing number of colonies
between 30-300 only are counted to eliminate
error.
 Calculate CFU/ml by multiplying number of
colonies by the dilution factor.
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B. SPREAD PLATE TECHNIQUE
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In this method different dilutions are done from the
bacterial suspension.
Sterile nutrient agar plates are prepared for each
dilution.
1 ml of each dilution is then poured on the agar
plates.
Using a glass or metal spreader- previously sterilized
by dipping it in alcohol and flaming-the bacterial
suspension is then uniformly spread on the plate.
The plates are left to dry and then incubated at 37oC
for 48 hours.
The plates containing number of colonies between 30300 only are counted to eliminate error.
Calculate CFU/ml by multiplying number of colonies
by the dilution factor.
SURFACE VIABLE TECHNIQUE
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Materials:
Culture of S. aureus.
Nutrient agar plate.
Ringer solution.
3 Test tubes.
Sterile 3ml pipettes.
Sterile 10ml pipette.
Sterile Pasteur pipette.
9 ml ringer solution
1ml
1ml
1ml bact.
Susp.
R.S
S
1
2
3
1:10
1:100
1:1000
dilution
1 drop in each
sector
Don’t Invert and incubate for 48h at
37℃
After incubation
Each sector from 1030 colonies
Count the colonies in each sector and record the results
RESULT

Count the number of colonies on each
sector in the plate which are in the range of
10-30.
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Over 30 reported as TNTC.
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Under 10 reported as TFTC.
RESULT
Sector number
1
2
3
4
# of colonies / sector
a
b
c
d
No of cell / 1ml of original culture (cfu/ml)= a+b+c+d X20X dilution
factor
4
SENSITIVITY TESTING
Lab # 6
SENSITIVITY TESTING
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Sensitivity testing is used to determine the
susceptibility of the microorganism to
various antimicrobial agents.
It can be done by :
Plate diffusion method using disc.
 Minimum inhibitory concentration [MIC].
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SENSITIVITY TESTING
 Antibiotic
sensitivity testing by the disc
diffusion method:
 Principle;
Rapid
 Accurate
 Inexpensive
The diameter of the resulting zones of
inhibition that surrounds a disc that has been
impregnated with a specific concentration of
the agent will be a measure of the
effectiveness of the antibiotics on the
microorganism.
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SENSITIVITY TESTING
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There are two types of antibiotic:Narrow spectrum
active against Gram +ve only or Gram –ve only
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Broad spectrum antibiotic
active against both types of bacteria
The
recommended medium →
Mueller Hinton agar
** The pH of the medium ( 7.2-7.4)
**5% defibrinated sheep blood is added to the
medium for certain fastidious organisms.
The
inoculum:
The turbidity of a broth culture / saline
suspension of the test organism has to
match a defined standard
“0.5 McFarland” (a barium sulphate
standard)
( the matched inoculum should give confluent
growth).
SENSITIVITY TESTING
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Materials:
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Cultures of E. coli.
Filter paper disc impregnated in antibiotic
solutions.
Different antibiotic solutions.
Muller Hinton agar plate.
Loop.
Forceps.
Procedure:
m.o
Az
45°c
24 h
at 37°c
Don’t invert
Aug
Cef
Az
Van
RESULTS:
Measure the diameter of each inhibition zone
* The diameter of the inhibition zones are
directly proportional to the susceptibility of
the microorganism to the antibiotics.

Van
Aug
Az
Cef
SENSITIVITY TESTING
 Results:
( in mm)
Mo
AB
1
2
3
4
E
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