1. PHT 226 Lab number 7 Total and viable count of bacteria

2. IMPORTANCE OF BACTERIAL COUNT V ARIOUS METHODS ARE USED IN MICROBIOLOGY TO MEASURE THE NUMBERS OF MICROORGANISMS PER UNIT VOLUME OF A GIVEN SAMPLE

3. 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.

4. H OW TO DETERMINE BACTERIAL GROWTH ? 1. D IRECT MICROSCOPIC COUNT : U SED TO DETERMINE THE NUMBER OF BOTH DEAD AND LIVING BACTERIAL CELLS ( HAEMOCYTOMETER ).

5. 2. Turbidimetric determination (viable & dead) I NCREASED TURBIDITY IN A CULTURE IS ANOTHER INDEX OF BACTERIAL GROWTH AND CELL NUMBERS. T HE INCREASE IN THE NUMBER OF CELLS DURING GROWTH INCREASES THE TURBIDITY. T URBIDIMETRIC DETERMINATION IS DONE USING A SPECTROPHOTOMETER.

6. 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.

8. How to USE THE SPECTROPHOTOMETER 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.

9. The amount of cells in the solution is directly proportional to the absorbance reading (linear relationship) 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. A graph of absorbance vs. concentration will give a straight line.

10. 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.

11. MICRO KJELDAHL METHOD MICRO KJELDAHL METHOD Bacterial cells are heated with conc H 2 SO 4 Ammonia is liberated. Ammonia is distilled & captured in boric acid solution. Titrate with 0.01N HCL Using methyl red- bromo cresol green as indicator.

12. 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. e.g; the growth of acid forming bacteria may be measured by simple titration of the culture using standard alkali.

13. 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. 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.

14. V IABLE 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.

15. 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 45 o C is poured in each plate with swirling. The plates are left to dry and then incubated at 37 o C 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.

16. B. S PREAD PLATE TECHNIQUE 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 37 o C 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.

17. S URFACE VIABLE TECHNIQUE 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.

18. 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

19. After incubation Each sector from 10- 30 colonies Count the colonies in each sector and record the results

20. R ESULT 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.

21. R ESULT 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

22. S ENSITIVITY T ESTING Lab # 6

23. S ENSITIVITY T ESTING 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].

24. S ENSITIVITY T ESTING 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.

25. S ENSITIVITY T ESTING 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

26. 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.

27. 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).

28. S ENSITIVITY T ESTING Materials: Cultures of E. coli. Filter paper disc impregnated in antibiotic solutions. Different antibiotic solutions. Muller Hinton agar plate. Loop. Forceps.

29. Procedure: 45°c 0.2ml m.o inoculate Aug AzVan Cef Az 24 h at 37°c incubate Don’t invert

30. R ESULTS : Measure the diameter of each inhibition zone 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. Cef Az Van Aug

31. S ENSITIVITY T ESTING Results: ( in mm) Mo MoAB E 1 2 3 4

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