Counting Microorganisms

Methods

• Turbidity measurements: Optical density• Viable counts• MPN• Direct counts

Turbidity measurements

• Measures the amount of light that can go through a sample

• The less the amount of light which goes through the sample the denser the population

• Mesures optical density or percent transmission

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Turbidity measurements

• Spectrophotometer (A600): – Measuring optical density

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Light

600nm

Detector….reading

Different reading

50

2.0

1.0

O.D. 600nm % Transmission

100

0

50

Cellular density

Inverse relationship

Turbidity measurements

Viable Counts  • Serial dilutions of sample

• Spread dilutions on an appropriate medium

• Each single colony originates from a colony forming unit (CFU)

• The number of colonies represents an approximation of the number of live bacteria in the sample

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Bacterial culture

CFU CFU CFU

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• 63 CFU/0.1ml of 10-5

• 630 CFU/1.0ml of 10-5

• 630 CFU/ml X 105 = 6.3 x 107/ml in original sampleWhat if there were 100 ml in the flask?

Serial Dilutions

• Advantages::– Gives a count of live microorganisms– Can differentiate between different microorganisms

• Limits:– No universal media

• Can’t ask how many bacteria in a lake• Can ask how many E. coli in a lake

– Requires growth– CFU one bacteria

• Ex. One CFU of Streptococcus one of E.coli=?

Viable Counts 

=?

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

Using a hemacytometer

Using a hemacytometer (Cont’d)

Using a hemacytometer (Cont’d)

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?

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 0.0013 2 1 150

Microscopy

Staining

Simple Staining

• Positive staining – Stains specimen– Staining independent of the species

• Negative staining– Staining of background– Staining independent of the species

Method

• Simple stain: – One stain– Allows to determine size, shape, and aggregation

of bacteria

Cell Shapes

• Coccus: – Spheres – Division along 1,2 or 3 axes– Division along different axes gives rise to different

aggregations– Types of aggregations are typical of different

bacterial genera

Cocci (Coccus)

Diplococcus

Streptococcus(4-20)

Tetrad

Staphylococcus

ArrangementsAxes of division

Hint: if name of genus ends in coccus, then the shape of the bacteria are cocci

Cell Shapes (Cont’d)

• Rods:– Division along one axis only– Types of aggregations are typical of different

bacterial genera

The Rods

Diplobacillus

Streptobacillus

ArrangementsAxes of division

Hint: if name of bacteria genus is Bacillus, then the shape of the bacteria are rods

If it doesn’t end in cocci, it’s probably a rod.

Microscopy

Differential Staining

Differential StainingGram Stain

• Divides bacteria into two groups

• Gram 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

Rules 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

Gram Positive Gram Negative

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

safraninVegetative cells(actively growing)

Spores(resistant

structures used for survival under

unfavourable conditions.)

Endospore(spore within

cell)

Sporangium(cell +

endospore)