THE METHODS FOR STUDYING MICROORGANISMS

Specimen collection: Nearly any object, common ones are body fluids and tissues, foods, water or soil. Specimens are removed by some form of sampling device. This may be a swab, syringe, or a inoculation needle and inoculation loop that holds, maintains and preserves the microbes in the sample.

The Five basic techniques to grow, examine and characterize

microorganisms INOCULATION INCUBATION ISOLATION INSPECTION IDENTIFICATION

Inoculation(producing a culture)

To cultivate or culture, one introduces a tiny sample(the inoculum)into a container of a nutrient medium which provides an environment in which they multiply

Selection of media with specialized functions can improve later steps of isolation and identification.

Incubation

To adjust the proper growth conditions of a sample

Promotes multiplication of the microbes over a period of hours, days and even weeks.

Produces a culture- the visible growth of the microbe in the medium

Isolation

The end result of inoculation and incubation in macroscopic form

The isolated microbes may take the form of separate colonies (discrete mounds of cells) on solid media, or turbidity in broths

Methods for isolating bacteria Streak method-a small droplet of culture or sample is

spread over the surface of the medium according to a pattern that gradually thins out the sample and separates the cells spatially over several sections of the plate

Loop Dilution/ pour plate-the sample is inoculated serially into a

series of cooled but still liquid agar tubes so as to dilute the number of cells in each successive tube in the series. Inoculated tubes are then plated out into sterile plates and are allowed to solidify. The end result is that the number of cells per volume is so decreased that cells have ample space to grow into separate colonies

Spread plate technique-A small volume of liquid diluted sample is

pipetted onto the surface of the medium and spread around evenly by a sterile tool (like a hockey stick).Like the streak method, cells are pushed into separate areas on the surface so that they can form individual colonies

Media: Providing Nutrients in the lab

3 Primary levels of media classification:1. Physical form2. Chemical composition3. Functional type

Physical state(medium’s normal consistency

Chemical composition( type of chemicals a medium contains)

Functional type(purpose of medium)

1. Liquid 2. Semisolid3. Solid(can be

converted into liquid

4. Solid (cannot be liquefied)

1. Synthetic (chemically defined)

2. Nonsynthetic (not chemically defined)

1. General purpose2. Enriched type3. Selective4. Differential5. Anaerobic growth6. Specimen

transport7. Assay8. enumeration

Physical states of media

Liquid media- are water based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted. These media, termed broths, milks or infusions, are made by dissolving various solutes in distilled water. Growth occurs throughout the container and can then present a dispersed, cloudy or particulate appearance.

Semisolid media- exhibit a clotlike consistency because they contain an amount of solidifying agent(agar or gelatin) that thickens them but does not produce a firm substrate. Semisolid media are used to to determine the motility of bacteria and to localize a reaction at a specific site.(0.3-0.5% agar) ex. SIM(hydrogen sulfide production and indole reaction)

Solid media- provide a firm surface on which cells can form discrete colonies and are advantageous in isolating and culturing bacteria and fungi.

2 forms1. Liquefiable- contain a solidifying agent that changes its

physical properties in response to temperature. Agar a complex polysaccharide isolated from the red alga Gelidium. It is solid at room temperature, and it melts at the boiling temperature of water (100). Ex. Nutient agar(beef extract and peptone, as well as 1.5% agar by weight)

2. Nonliquefiable –they do not melt. They include materials such as rice grains (to grow fungi), cooked meat media(anaerobes), and potato slices

Chemical content of Media

Synthetic- contain pure organic and inorganic compounds

Nonsynthetic/compex media-contain at least one ingredients that are not chemically definable- extracts of animals, plants or yeasts, ground up cells, tissues and secretions, milk and peptone . Blood agar, nutrient broth and MacConkey agar.

Media to suit Every Function

1. General purpose media- are designed to grow as broad spectrum of microbes as possible. As a rule, they are nonsynthetic and contain a mixture of nutrients that could support pathogens and nonpathogens alike. Ex. BHI, TSA

2. Enriched medium- conains complex organic substances such as blood serum, hemoglobin or special growth factors that certain species must have inorder to grow. Bacteria that require growth factors and complex nutrients are fastidius. Ex. Blood agar to grow fastidius streptococci and other pathogens. Thayer Martin medium- for neisseria

3. Selective medium- contains one or mre agents that inhibit one or more agents that inhibit the growth of a certain microbe or microbes but not others and thereby selects or encourages microbe nd allow it grow.MSA contains a concentration of NaCl that is quite inhibitory to most human pathogens except staphyloccoccus.

Selective media, agents and functions

Medium Selective agent functions

Mueller telluriteEnterococcus faecalis brothPhenylethanol agarTomato juice agarMacConkey agarSalmonella/shigella agarLowenstein-JensenSabourauds agar

Potassium telluriteSodium azide tetrazoliumPhenylethanol chlorideTomato juice, acidBile, crystal violetBile, citrate, brilliant greenMalachite green dyePh 5.6(acid)

C. diptheriaeFecal enterococciStaph and StrepLactobacilli from salivaGram negative entericsSalmonella and shigellaMycobacteriaFungi, inhibits bacteria

Differential media

Grow several types of microorganisms and are designed to display visible differences among those microorganisms. Differentiation shows up as variations in colony size or color, in media color changes or in the formation of gas bubbles and precipitates

medium Substances that facilitatedifferentiation

Differentiates between

Blood agar Intact red blood cellsManitol,phenol and 7.5%

Types of hemolysisSpecies of stap. NaCl

Hektoen enteric agar

Spirit blue agar

Urea broth

Sulfur indole motilityTriple sugar iron gar

Nacl

Bromthymol blue, acid fushin,sucrose, salicin, thiosulfate, ferric ammonium citrate, and bile

Spirit blue dye and oil

Urea, phenol red,

thiosulfate, ironTriple sugars, iron and phenol red dye

Also inhibits the salt-sensitive speciesSalmonell, shigella and othe lactose fermenters and nonfermentersDyes and bile also inhibit gram positive bacteriaBacteria that use fats from those that do notBacteria that hydrolyze urea to ammoniaH2S gas producers from nonproducersFermentation of sugars, H2S production

medium Substances that facilitate differentiation

Differentiates between

XLD agar

Birdseed agar

Lysine, xylose, iron, thiosulfate,phenol red

Seeds from thistle plant

Enterobacter, escherichia, proteus, providencia, salmonella and shigellaCryptococcus neoformans and other fungi

Miscellaneous media

A reducing medium contains substance(cystine or thioglycollic acid) that absorbs oxygen or slows the penetration of oxygen in a medium, thus reducing its availability. Reducing media are important for growing anaerobic bacteria or determining oxygen requirements.

Transport media

Are used to maintain and preserve specimens that have to be held for a period of time for clinical analysis or to sustain delicate species that die rpidly if not held under stable conditions.

Assay media

To test effectiveness of antimicrobial drugs and by drug manufacturers to assess the effect of disinfectants, antiseptics, cosmetics and preservatives on the growth of microorganisms

Enumeration media

Used by industrial and environmental microbiologists to count the numbers of organisms in milk, water, food, soil and other samples

A culture may exist in one of the following forms:

1. Pure2. Mixed3. contaminated

Inspection

Cultures are observed macroscopically for obvious growth characteristics(color, texture,size) that coild be useful in analyzing specimen contents. Slides are made to assess microscopic details such as shapes, size, and motility. Staining techniques may be used to gather specific information on microscopic morphology

Identification

A major purpose of the 5 I’s is to determine the type of microbe, usually to the level of species. Specialized tests include biochemical tests to determine metabolic activities specific to the microbe.