PHT 226Lab number 7

Total and viable count of bacteria

IMPORTANCE OF BACTERIAL IMPORTANCE OF BACTERIAL COUNTCOUNT

VARIOUS METHODS ARE USED VARIOUS METHODS ARE USED IN MICROBIOLOGY TO IN MICROBIOLOGY TO MEASURE THE NUMBERS OF MEASURE THE NUMBERS OF MICROORGANISMS PER UNIT MICROORGANISMS PER UNIT VOLUME OF A GIVEN SAMPLEVOLUME OF A GIVEN SAMPLE

This measurement is needed This measurement is needed for:-for:-

1.1. Standardization of inocula in Standardization of inocula in microbiological assay microbiological assay

[e.g. evaluation of [e.g. evaluation of antimicrobial agents, assay antimicrobial agents, assay of vitamins]of vitamins]

1.1. Industrial fermentation.Industrial fermentation.2.2. Evaluation of sterilization Evaluation of sterilization

technique. technique.

HOW TO DETERMINE BACTERIAL HOW TO DETERMINE BACTERIAL GROWTH?GROWTH?

1. 1. DIRECT MICROSCOPIC COUNT:DIRECT MICROSCOPIC COUNT: USED TO DETERMINE THE USED TO DETERMINE THE NUMBER OF NUMBER OF BOTH DEAD AND BOTH DEAD AND LIVING BACTERIAL CELLSLIVING BACTERIAL CELLS (HAEMOCYTOMETER(HAEMOCYTOMETER))..

2. 2. Turbidimetric determination Turbidimetric determination (viable & dead)(viable & dead)

INCREASED TURBIDITY IN A CULTURE IS INCREASED TURBIDITY IN A CULTURE IS ANOTHER INDEX OF BACTERIAL GROWTH ANOTHER INDEX OF BACTERIAL GROWTH AND CELL NUMBERS.AND CELL NUMBERS.

THE INCREASE IN THE NUMBER OF CELLS THE INCREASE IN THE NUMBER OF CELLS DURING GROWTH INCREASES THE DURING GROWTH INCREASES THE TURBIDITY.TURBIDITY.

TURBIDIMETRIC DETERMINATION IS DONE TURBIDIMETRIC DETERMINATION IS DONE

USING A USING A SPECTROPHOTOMETER.SPECTROPHOTOMETER.

SPECTROPHOTOMETER Measure growth rates with a

spectrophotometer

Direct relationship between cell number and absorbance, Otherwise known as Optical DensityMore bacteria= higher

absorbance.less light reaches sensorCells 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 SPECTROPHOTOMETERHow 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.

The amount of cells The amount of cells in the solution is in the solution is directly directly proportional to the proportional to the absorbance readingabsorbance reading (linear (linear relationship) relationship) 

A graph of A graph of absorbance vs. absorbance vs. concentration will concentration will give a straight line.give a straight line.

33. . Dry weight and nitrogen Dry weight and nitrogen content determinations:content determinations:

In this method, the bacterial In this method, the bacterial

cells are collected by cells are collected by centrifugation, then dried in an centrifugation, then dried in an oven overnight at 85oven overnight at 85℃. ℃.

The dry weight of bacterial mass The dry weight of bacterial mass will be proportional to their will be proportional to their number. number.

Also the nitrogen content of the Also the nitrogen content of the dry sample can be determined dry sample can be determined by micro kjeldahl method.by micro kjeldahl method.

MICRO KJELDAHL MICRO KJELDAHL METHODMETHOD

Bacterial cells are heated with Bacterial cells are heated with conc Hconc H22SOSO44

Ammonia is liberated.Ammonia is liberated.Ammonia is distilled & captured in Ammonia is distilled & captured in

boric acid solution.boric acid solution.Titrate with 0.01N HCLTitrate with 0.01N HCLUsing methyl red- bromo cresol Using methyl red- bromo cresol

green as indicator.green as indicator.

4. Measurement of 4. Measurement of microbial activity:microbial activity:

Many microbial activity measured Many microbial activity measured quantitatively and used as a quantitatively and used as a measure of microbial growth. measure of microbial growth. e.g; the growth of acid forming e.g; the growth of acid forming bacteria may be measured by bacteria may be measured by simple titration of the culture simple titration of the culture using standard alkali.using standard alkali.

5. Viable count of 5. Viable count of bacteria:bacteria:

in this method in this method only viable cellsonly viable cells which are capable of reproduction which are capable of reproduction are counted.are counted.

Principle:Principle:

Based on the fact that if the Based on the fact that if the viable cells are allowed to grow viable cells are allowed to grow apart from each other on a solid apart from each other on a solid medium, each cell develops into medium, each cell develops into one visible colony. The number of one visible colony. The number of colonies obtained is equal to the colonies obtained is equal to the number of viable cells. number of viable cells.

VIABLE COUNT OF VIABLE COUNT OF BACTERIABACTERIA There are different procedures used There are different procedures used

to determine viable bacterial count:-to determine viable bacterial count:-

a. Pour plate method.a. Pour plate method.

b. Spread plate method.b. Spread plate method.

c. Surface viable count.c. Surface viable count.

A. POUR PLATE METHODA. 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.

B. SPREAD PLATE B. SPREAD PLATE TECHNIQUETECHNIQUE 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 30-300 only are counted to eliminate error.

Calculate CFU/ml by multiplying number of colonies by the dilution factor.

SURFACE VIABLE SURFACE VIABLE TECHNIQUETECHNIQUE

Materials:Materials: Culture of S. aureus.Culture of S. aureus. Nutrient agar plate.Nutrient agar plate. Ringer solution.Ringer solution. 3 Test tubes.3 Test tubes. Sterile 3ml pipettes.Sterile 3ml pipettes. Sterile 10ml pipette.Sterile 10ml pipette. Sterile Pasteur pipette.Sterile Pasteur pipette.

1 2 3

9 ml ringer solution

S

1ml bact. Susp.

1:10

1ml

1:100

1ml

1:1000 dilution

Don’t Invert and incubate for 48h at 37℃

R.S

1 drop in each sector

After incubation

Each sector from 10-30 colonies

Count the colonies in each sector and record the results

RESULTRESULT

Count the number of colonies on each sector in the plate which are in the range of 10-30.

Over 30 reported as TNTC.

Under 10 reported as TFTC.

RESULTRESULT

Sector number # of colonies/ sector

1 a

2 b

3 c

4 d

No of cell / 1ml of original culture (cfu/ml)= a+b+c+d X20X dilution factor 4

SENSITIVITY TESTINGLab # 6

SENSITIVITY TESTING

Sensitivity testing is used Sensitivity testing is used to to determine determine the susceptibility of the microorganism the susceptibility of the microorganism to various antimicrobial agents.to various antimicrobial agents.

It can be done by : Plate diffusion method using disc. Minimum inhibitory concentration [MIC].

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.

SENSITIVITY TESTING

There are two types of antibiotic:-

Narrow spectrum active against Gram +ve only or Gram –ve

only

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

Materials: Cultures of E. coli. Filter paper disc impregnated in antibiotic

solutions. Different antibiotic solutions. Muller Hinton agar plate. Loop. Forceps.

Procedure:

45°c

0.2ml

m.o

inoculate Aug

Az Van

Cef

Az

24 h

at 37°c

incubate

Don’t invertDon’t invert

RESULTS: Measure the diameter of each inhibition zoneMeasure the diameter of each inhibition zone

* The diameter of the inhibition zones are * The diameter of the inhibition zones are directly proportional to the susceptibility of directly proportional to the susceptibility of the microorganism to the antibiotics.the microorganism to the antibiotics.

Cef

Az

VanAug

SENSITIVITY TESTING

Results: ( in mm)

MoMo

ABAB EE

11

22

33

4 4

THANK YOU