The 5 I’s of culturing microbes Inoculation – introduction of a sample into a container of media Incubation – under conditions that allow growth Isolation –separating one species from another Inspection Identification

Fig. 3.1a

Fig. 3.1b

Fig. 3.1c

Isolation If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony A colony consists of one species

Isolation technique

Fig. 3.3

Fig. 3.1d

Media – providing nutrients in the laboratory Most commonly used: nutrient broth – liquid medium containing beef extract & peptone nutrient agar – solid media containing beef extract, peptone & agar agar is a complex polysaccharide isolated from red algae solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC provides framework to hold moisture & nutrients not digestible for most microbes

Types of media synthetic – contains pure organic & inorganic compounds in an exact chemical formula complex or nonsynthetic – contains at least one ingredient that is not chemically definable general purpose media- grows a broad range of microbes, usually nonsynthetic enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

Enriched media Blood agar Chocolate agar

selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

Selective media Mannitol salt agar MacConkey agar

Differential media TSIA CHROMagar Orientation

Miscellaneous media reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria carbohydrate fermentation medium- contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi transport media

Carbohydrate fermentation media

Fig. 3.12

Cultures Pure culture- only grows one type of microbe Mixed culture- contains 2 or more microbes Contaninated culture- was once pure, but unwanted microbes became introduced Axenic- free of other living things except the one being studied

Maintenance and disposal of cultures Catalogues of microbes are maintained at the American Type Culture Collection Microbes are disposed of by various forms of sterilization

Why use animals? You can test the effects of drugs, etc without risking the lives of humans To evaluate disease, or study the process and cause of disease Source of antibodies, antitoxins May be used to determine the pathogenicity or toxicity of certain bacteria To grow microbes which cannot be grown on artificial media

compound light microscope

magnification – ability to enlarge objects resolving power – ability to show detail refraction- bending ability of light

Pathway of light

Total Magnification Ocular power Objective power Total magnification 10x 4x Scanning 40x Low 100x High and dry 400x Oil immersion 1000x

resolution The human eye can only resolve objects about 0.2mm apart Oil immersion allows resolution to about 0.2um RP= wavelength of light (nm)/2 x NA NA is the cone of light entering the lens

Effect of wavelength on resolution

Oil immersion lens

Other constraints for a clear image Other things needed for a clear image are Quality of the lens Quality of the light source Lack of contrast in the specimen

Effect of magnification

Types of light microscopes Bright-field – most widely used, specimen is darker than surrounding field Dark-field – brightly illuminated specimens surrounded by dark field Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

3 views of a cell Brightfield 400x Darkfield 400x Phase contrast 400x

Types of light microscopes Differential interference contrast- uses light waves and 2 prisms which produce contrasting colors; well defined, colored, 3D images

Phase contrast 600x Fig. 3.21 DIC 160x

Fluorescence Microscope Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye Uses dyes that emit visible light when bombarded with shorter uv rays. Useful in diagnosing infections

Cheek cells and streptococci

Light Microscopes Confocal- uses a laser to scan sections of the specimen

Electron microscopy Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds. Electron waves are 100,000X shorter than the waves of visible light. Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength. Magnification between 5,000X and 1,000,000X

2 types of electron microscopes Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

Scanning Electron Micrograph paramecium

Table 3.6

Table 3.5

Specimen Preparation The way a slide is prepared depends upon The condition of the specimen The needs of the examiner The type of microscope available

Specimen preparation wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement Wet mount- a drop of culture is placed on a slide with a cover glass Quick and easy to prepare Cover glass may damage larger cells Slide is susceptible to drying and can contaiminate the handler’s fingers

Specimen preparation Hanging drop- uses a special slide with a depression; sample is placed in the depression and vaseline is placed around it; coverslip overlies the sample

Specimen preparation fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts. Kills the microbe Allows adherence to the slide Preserves cellular components in a natural state with minimal distortion

Staining cationic dyes - basic, with positive charges on the chromophore anionic dyes - acidic, with negative charges on the chromophore surfaces of microbes are negatively charged and attract basic dyes – positive staining. negative staining – microbe repels dye & it stains the background Simple Reduced shrinkage or distortion of cells Can observe capsules

Staining simple stains –one dye is used differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain special stains: capsule and flagellar stains

Table 3.7