1. Counting Microorganisms

2. Methods Turbidity measurements Viable counts Most probable number Direct counts

3. Most probable Number: MPN –Based on Probability Statistics –Presumptive test based on given characteristics –Broth Technique

4. Most Probable Number (MPN) Begin with Broth to detect desired characteristic Inoculate different dilutions of sample to be tested in each of three tubes -1 -2 -3 -4 -5 -6 Dilution 3 Tubes/Dilution 1 ml of Each Dilution into Each Tube After suitable incubation period, record POSITIVE TUBES (Have GROWTH and desired characteristics)

5. MPN - Continued Objective is to “DILUTE OUT” the organism to zero Following the incubation, the number of tubes showing the desired characteristics are recorded Example of results for a suspension of 1g/10 ml of soil Dilutions: -1 -2 -3 -4 Positive tubes: 3 2 1 0 –Choose correct sequence: 321 and look up in table –Multiply result by middle dilution factor »150 X 10 2 = 1.5 X 10 4 /mL »Since you have 1g in 10mL must multiply again by 10 »1.5 X 10 5 /g Pos. tubes MPN/g (mL) 0.100.010.001 321150

6. Direct Counts The sample to be counted is applied onto a hemacytometer slide that holds a fixed volume in a counting chamber The number of cells is counted in several independent squares on the slide’s grid The number of cells in the given volume is then calculated

7. Determining the Direct Count 7 Count the number of cells in three independent squares –8, 8 and 5 Determine the mean –(8 + 8 + 5)/3 =7 –Therefore 7 cells/square

8. Determining the Direct Count (Cont’d) 8 Calculate the volume of a square: = 0.1cm X 0.1cm X 0.01cm= 1 X 10 -4 cm 3 or ml Divide the average number of cells by the the volume of a square –Therefore 7/ 1 X 10 -4 ml = 7 X 10 4 cells/ml 1mm Depth: 0.1mm

9. Problem A 500μl sample is applied to a hemacytometer slide with the following dimensions: 0.1mm X 0.1mm X 0.02mm. Counts of 6, 4 and 2 cells were obtained from three independent squares. What was the number of cells per milliliter in the original sample if the counting chamber possesses 100 squares?

10. Microscopy Differential Staining

11. Differential Staining Gram Stain Divides bacteria into two groups Gram Negative & Gram Positive

12. Stained Purple –Rods Genera Bacillus and Clostridium –Coccus Genera Streptococcus, Staphylococcus and Micrococcus

13. Gram Negative Stained Red –Rods: Genera Escherichia, Salmonella, Proteus, etc. –Coccus: Genera Neisseria, Moraxella and Acinetobacter

14. Rule of thumb If the genus is Bacillus or Clostridium = Gram (+) rod If the genus name ends in coccus or cocci (besides 3 exceptions, which are Gram (-)) = coccus shape and Gram (+) If not part of the rules above, = Gram (-) rods

15. Cell Wall 15 Peptidoglycan wall Plasma Membrane Lipopolysaccharide layer Absent Gram + Vs Gram -

16. Method – Primary staining 1.Staining with crystal violet 2.Addition of Gram’s iodine (Mordant) + Gram positiveGram negative - - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS ++++++++++++++

17. Method – Differential step 3.Alcohol wash Gram positiveGram negative - - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall: peptidoglycan LPS +++++++ +++++++ Wall is dehydrated – Stain + iodine complex is trapped Wall is not dehydrated – Complex is not trapped

18. Method – Counter Stain 4.Staining with Safranin Gram positiveGram negative - - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS +++++++ + ++++++++++++++ 18

19. Summary 19 Fixation Primary staining Crystal violet Counter staining Safranin Wash Destaining

20. Acid Fast Staining Diagnostic staining of Mycobacterium –Pathogens associated with Tuberculosis and Leprosy –Cell wall has mycoic acid Waxy, very impermeable

21. Method Basis: –High level of compounds similar to waxes in their cell walls, Mycoic acid, makes these bacteria resistant to traditional staining techniques

22. Method (Cont’d) Cell wall is permeabilized with heat Staining with basic fuchsine –Phenol based, soluble in mycoic layer –Cooling returns cell wall to its impermeable state Stain is trapped Wash with acid alcohol –Differential step Mycobacteria retain stain Other bacteria lose the stain

23. Spore Stain Spores: –Differentiated bacterial cell –Resistant to heat, desiccation, ultraviolet, and different chemical treatments Thus very resistant to staining too! –Typical of Gram positive rods Genera Bacillus and Clostridium –Unfavorable conditions induce sporogenesis Differentiation of vegetative cell to endospore –E.g. Anthrax

24. Malachite Green Staining Permeabilization of spores with heat Primary staining with malachite green Wash Counter staining with safranin Vegetative cells (actively growing) Spores (resistant structures used for survival under unfavourable conditions.) Endospore (spore within cell) Sporangium (cell + endospore)

25. Pathogens

26. 19 th Century: Robert Koch Studies anthrax disease which kills cows Grows in pure culture bacteria obtained from the blood of diseased animals –Bacillus anthracis Observations: –Blood of diseased animals transmits the disease –The microorganisms is found only in diseased animals –The microorganism grown in the lab transmits the disease to healthy animals 26

27. Robert Koch (Cont’d) Conclusion: Microorganisms are responsible of diseases –Pathogens These results lead Robert Koch to formulate guidelines to associate a microorganism to a disease –Koch’s postulates 27

28. Koch’s Postulates The microorganism must be present in each diseased case but absent from healthy individuals The microorganism must be isolated and grown in pure cultures The disease must develop when the isolated microorganism is inoculated in a healthy host The same microorganism must be isolated again from the diseased host 28