Counting Microorganisms

Methods

• Turbidity measurements• Viable counts• Most probable number• Direct counts

Most probable Number: MPN

– Based on Probability Statistics– Presumptive test based on given characteristics– Broth Technique

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 -6Dilution

3 Tubes/Dilution

1 ml of Each Dilution into Each Tube

After suitable incubation period, record POSITIVE TUBES (Have GROWTH and desired characteristics)

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 102 = 1.5 X 104/mL» Since you have 1g in 10mL must multiply again by 10» 1.5 X 105/g

Pos. tubesMPN/g (mL)

0.10 0.01 0.001

3 2 1 150

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

Determining the Direct Count

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• 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

Determining the Direct Count (Cont’d)

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• Calculate the volume of a square:= 0.1cm X 0.1cm X 0.01cm= 1 X 10-4cm3 or ml

• Divide the average number of cells by the the volume of a square– Therefore 7/ 1 X 10-4 ml = 7 X 104 cells/ml

1mm

1mmDepth: 0.1mm

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?

Microscopy

Differential Staining

Differential StainingGram Stain

Divides bacteria into two groupsGram Negative & Gram Positive

• Stained Purple – Rods

• Genera Bacillus and Clostridium– Coccus

• Genera Streptococcus, Staphylococcus and Micrococcus

Gram Negative

• Stained Red– Rods:

• Genera Escherichia, Salmonella, Proteus, etc.

– Coccus: • Genera Neisseria, Moraxella and Acinetobacter

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

Cell Wall

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Peptidoglycanwall

PlasmaMembrane

Lipopolysaccharidelayer

Absent

Gram + Vs Gram -

Method – Primary staining

1. Staining with crystal violet2. Addition of Gram’s iodine (Mordant)

+

Gram positive Gram negative

- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS

Method – Differential step

3. Alcohol wash

Gram positive Gram negative

- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall: peptidoglycan LPS

+ + + + + + + + + + + + + +

Wall is dehydrated – Stain + iodine complex is trapped

Wall is not dehydrated – Complex is not trapped

Method – Counter Stain

4. Staining with Safranin

Gram positive Gram negative

- - - - - - - - - - - - - - - Plasma membrane - - - - - - - - - - - - - - - Wall:peptidoglycan LPS

+ + + + + + +

+

+ + + + + + + + + + + + + +

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Summary

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Fixation

Primary stainingCrystal violet

Counter stainingSafranin

WashDestaining

Acid Fast Staining

• Diagnostic staining of Mycobacterium– Pathogens associated with Tuberculosis and Leprosy– Cell wall has mycoic acid

• Waxy, very impermeable

Method

• Basis: – High level of compounds similar to waxes in their

cell walls, Mycoic acid, makes these bacteria resistant to traditional staining techniques

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

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

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)

Pathogens

19th 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 animals26

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

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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

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