The Study of Microorganisms Microbiology The Study of Microorganisms

Definition of a Microorganism Derived from the Greek: Mikros, «small» and Organismos, “organism” Microscopic organism which is single celled (unicellular) or a mass of identical (undifferentiated) cells Includes bacteria, fungi, algae, viruses, and protozoans

Microorganisms in the Lab Growth Media

Goals Growth under controlled conditions Maintenance Isolation of pure cultures Metabolic testing

Types Liquid (Broths) Solid media Allows growth in suspension Uniform distribution of nutrients, environmental parameters and others Allows growth of large volumes Solid media Same as liquid media + solidification agent Agar: Polysaccharide derived from an algae

Growth in Broths Non inoculated clear Turbid + sediment Turbid Clear + sediment

Growth on Agar Growth on solid surface Isolated growth Allows isolation of single colonies Allows isolation of pure cultures Easier for counting colonies and observing morphology of colonies. Plates are used to: grow and culture bacteria, fungi, animal tissues, or plant tissues obtain separated pure cultures of bacteria (plate streaking) count colonies from serial dilutions test for growth and reactions on certain materials (such as manitol-salt, or blood agar) test for bacteria viruses (bacteriophages) test for resistance to materials (such as antibiotics) or nutritional needs (this is also used to select for bacteria with certain properties or favor the growth of one type/strain over others) Single colony

Solid Media (Cont’d) Slants Stab Growth on surface and in depth Different availabilities of oxygen Long term storage Stab Semi-solid medium Low availability of oxygen

Counting Microorganisms

Methods Turbidity measurements: Optical density Direct counts Viable counts MPN

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

Turbidity measurements Spectrophotometer (A600): Measuring optical density Light 600nm Detector….reading Different reading

Turbidity measurements 2.0 1.0 O.D. 600nm % Transmission 100 50 Cellular density Inverse relationship

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

Direct Counts Advantages: Limits: Quick Growth is not required No information about organism required Limits: Does not discriminate between live and dead May be difficult to distinguish bacteria from detritus

Using a hemacytometer

Using a hemacytometer (Cont’d)

Hemacytometer This slide has 2 independent counting chambers

Using a hemacytometer (Cont’d)

Determining the Direct Count 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) 1mm Depth: 0.1mm 1mm 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

Problem A 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? 2x10^9 cells/ml ...100 squares is relevant. Recall for cell culture: 1mmx1mmx0.1mm =0.1x0.1x0.01cm =0.0001cm3 10^-4 10^4 dil factor

Viable Counts A viable cell: a cell which is able to divide and form a population (or colony) A viable cell count is done by diluting the original sample Plating aliquots of the dilutions onto an appropriate culture medium Incubating the plates under appropriate conditions to allow growth Colonies are formed Colonies are counted and original number of viable cells is calculated according to the dilution used

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

Dilution of Bacterial Sample

Dilution of Bacterial Sample

Dilution of Bacterial Sample

Dilution of Bacterial Sample

Plating of Diluted Samples 5672 57 4

Viable Counts The total number of viable cells is reported as Colony-Forming Units (CFUs) rather than cell numbers Each single colony originates from a colony forming unit (CFU) A plate having 30-300 colonies is chosen Calculation: Number of colonies on plate X reciprocal of dilution (dilution factor) = Number of CFU/mL Ex. 57 CFU/0.1mL X 106 = 5.7 X 107 CFU/mL

Serial Dilutions 63 CFU/0.1ml of 10-5 630 CFU/1.0ml of 10-5 Bacterial culture CFU 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 sample What if there were 100 ml in the flask?

= = Viable Counts Advantages: Limits: 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 Only living cells develop colonies Clumps or chains of cells develop into a single colony CFU one bacteria Ex. One CFU of Streptococcus  one of E.coli Can differentiate between different microorganisms = b/c they are single colonies can count how many of each distinct morphology, colour etc. No universal media = if you have a mix of bacteria, not all will be happy on one media type. Requires growth = if you have live, but non reproducing bacteria, they won’t show up. = i.e. recall bacteriostatic antibiotics. = ? = ?

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 -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)

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. tubes MPN/g (mL) 0.10 0.01 0.001 3 2 1 150

Microscopy Staining

Simple Staining Positive staining Negative staining Stains specimen Staining independent of the species Negative staining Staining of background

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) Axes of division Arrangements Diplococcus Streptococcus (4-20) Tetrad Staphylococcus 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

If it doesn’t end in cocci, it’s probably a rod. The Rods Axes of division Arrangements Diplobacillus Streptobacillus 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.

Differential Staining Microscopy Differential Staining

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

Gram Positives Stained Purple Rods Coccus Genera Bacillus and Clostridium Coccus Genera Streptococcus, Staphylococcus and Micrococcus -Rule of thumb: cocci and not Neisseria, Moraxella and Acinetobacter = G+ cocci (sphere) -Genus is Bacillus or Clostridium then = rod G+ -All others are rod G-

Gram Negative Stained Red Rods: Coccus: 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

Gram Staining- Principal Uses a combination of two stains Primary stain - Crystal violet Purple Secondary stain – Safranin Red Gram positive The cell wall traps the 1o stain Gram negative Cell wall does not allow 1o stain to be trapped

Cell Wall

Method – Primary Stain Staining with crystal violet + Staining with crystal violet Add Gram’s iodine (Mordant) + + - - - - - - - - - - - - - - - Wall: peptidoglycan LPS Plasma membrane - - - - - - - - - - - - - - - Gram positive Gram negative

Method – Differential Step Alcohol wash Cell wall is dehydrated and less permeable – Stai + iodine complex is trapped LPS layer is dissolved Cell wall is dehydrated, but permeable – Complex is not trapped Wall: peptidoglycan LPS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Plasma membrane + + Gram positive Gram Negative

Method – Counter Stain Staining with Safranin + + + Wall: peptidoglycan LPS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Plasma membrane + Gram positive Gram Negative

Summary Fixation Primary stain Crystal violet Wash Decolorization Counter stain Safranin

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