Practical 3 Bacteria Culture and Isolation Techniques PDF

PRACTICAL 3 BACTERIAL CULTURES AND ISOLATION TECHNIQUES.  The purpose of using cultural techniques in microbiology is to demonstrate the presence of organisms which may cause disease, and when indicated, to test the sensitivity of pathogens to antimicrobial agents. General and specialized media...

PRACTICAL 3 BACTERIAL CULTURES AND ISOLATION TECHNIQUES.  The purpose of using cultural techniques in microbiology is to demonstrate the presence of organisms which may cause disease, and when indicated, to test the sensitivity of pathogens to antimicrobial agents. General and specialized media are required for bacterial growth and characterization, the culture media contain nutritional and physical requirement for growths includes;  Water  Source of energy  Sources of carbon, nitrogen ,sulfur, phosphorus etc  Minerals  Vitamins and growth factors. There are different types of culture media and they are classified into;  Basic  Enriched  Selective  Indicator  Transport  Identification.  A: BASIC MEDIA These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms that do not have special nutritional requirements. Purpose: 1: used to prepare enriched media 2: To maintain stock cultures for control strains of bacteria 3. Used for sub culturing strains which are used in performing biochemical test and serological identification tests. B: ENRICHED MEDIA: These are media required for growth of organisms with exacting growth requirements such as H.influenzae, Neiserria spps. The basic media can be enriched with whole / lysed blood, serum. Peptone, yeast extracts, vitamins and other growth factor.  Purpose: used to promote growth especially on samples collected in sterile sites to ensure rapid multiplication of pathogens which may be present only in small numbers. C: SELECTIVE MEDIA These are solid media which contain substances (e g.bile salts or chemicals ,dyes ,antibiotics) which inhibit the growth of one organisms to allow the growth of another to be more clearly demonstrated. Purpose: used for culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants overgrowing a pathogens media incorporating antibiotics are usually expensive. N.B: OTHER WAYS TO SELECT ORGANISMS Incubation conditions may be used to select microorganisms eg.P.aeruginosa is inhibited by anaerobic conditions.Also Ph of the medium may make it selective for particular organisms e.g ;V. cholera are isolated in TCBS Agar. Also temperature may also help to grow at 4c whereas other organisms are inhibited.Growth ,however, is slow. Image of Chrome agar Strep B, both a selective and differential media.  D. INDICATOR (DIFFERENTIAL) MEDIA: These re media which dyes or other substances are added to differentiate microorganisms Purposes :Differentiate bacteria by incorporating an indicator which change colour when acid is produced following fermentation of specific carbohydrates e.g. MacConkey agar. Note: many media used to isolate pathogens are both selective and enrichment/both selective and differential.  E.TRANSPORT MEDIA These are media used to prevent overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection from health centers to District/regional microbiology laboratories.EG Carry-Blair medium for enteric pathogens and for Amies transport medium to ensure survival of gonococci. SOLID,SEMISOLID AND FLUID CULTURE MEDIA Culture can be classified by consistency as: I/SOLID-It is incorporated by a gelling agent such as agar  Agar is polysaccharides extract obtained from seaweed, it is used to solidify culture media at 1-2% w/v it melt at 90-95 but solidify at 45c.solid media are prepared in Petri dishes and tubes/bottles as stab/ deep or as slope/slant 2/Semi-solid culture media This form of medium contains a small amount of agar 0.4-0.5%w/v to fluid medium they mainly used as transport medium. For motility and biochemical tests. 3. FLUID CULTURE MEDIA It has no agar and it is used to produce surface growth on medium eg;vibrio cholera in Alkaline peptone water. It also used in biochemical tests such as peptone water sugars containing tryptophan containing tryptophan for indole production by some enterobactericea. CHOICE OF CULTURE MEDIA The choice of media depend on the following factors  The major pathogens to be isolated and their growth requirements  Whether the sampling site have normal flora or not.  Cost,availabilitynand stability of differential media.  Training and experience of laboratory personell. PREPARATION OF SOLID MEDIA A solid media contains 1 to 2% agar. An agar is complex carbohydrate extracted from marine algae that solidifies below temperature of 45C.It is not a nutritional component.  From the media bottle, in the “direction for use”, read the concentration given e.g.Dissolve 40g in 1 litre.  Calculate the volume you need to prepare according to samples and sterile plates that you have e.g. If you have 25 plates each carrying 20ml of media then the total volume will be 25x20=500mls  Then calculate the mount of media to be measured on balance. 40g…………………………………………………………..1000ml x………………………………………………………………500mls X=20g  After measuring the powder dissolve in distilled water and then boil to dissolve completely as instructed in “direction for use”  If the autoclaving is recommended then autoclave the media (steam sterilization) at 121°C, 15lbs pressure for 15minutes.  Cool the media to about 45°C before pouring it into the petri dishes. Remember when pouring the media the flame should be very close to minimize contamination. Immediately after pouring flame the surface of media to minimize contamination and remove air bubbles that may bring confusion with the bacteria colony. NOTE: For liquid media the preparation and measurement procedure as well as sterilization is the same EXCEPT that in liquid media the agar is not there and it doesn’t solidify so media is poured in bottles. PREPARATION OF AGAR SLANT An agar slant consists of agar based medium in a culture tube.It is called slant because the tube is placed at an angle during cooling to give a slanted surface for inoculation. It is used for stock pure culture and biochemical tests. E.g Triple sugar iron agar(TSI). EXERCISE Experiment 3.1 Prepare the basic/general used media I.e MacConkey agar ,nutrient agar and Blood agar. PRACTICAL 3.2 Cultivation of microorganisms from various samples OBJECTIVES OF THE PRACTICAL  To familiaze the streaking techniques  To observe different colonies of microorganisms and appreciate the need of isolation to obtain the pure culture.  Familiarize the labeling procedure Materials 1.Samples : urine, tape water, saliva, puss swabs, vaginal swabs, broth culture. 2. Wire loop 3.Source of flame e.g Bunsen burner 4. Solid media (in petri dishes)& broth media (liquid media in tubes). Procedures  For liquid sample, sterilize a wire loop on flame  Leave to cool, deep the wire loop into the sample and streak it as follows:  Sterilize the wire loop before you do for the next sample  For solid sample or powder, dissolve the sample in little amount of sterile normal saline and then inoculation procedures can follow as above.  Place the plates at incubator at 37°C for overnight.  If the anaerobic or microaerophilic microorganisms are suspected then place the cultured plates in anaerobic jar and candle jar respectively before placing them in the incubator. NOTE :The growth temperature of incubator is regulated depending on growth characteristics. MACROMORHOLOGY OF MICROORGANISM  To study and familiarize the macro morphology of different colonies from different bacterial cultures.  Study bacterial reaction to different media E.g macConkey and blood agar. Materials: Pure culture of staphylococcus spp, Klebsiella & E.coli. Procedures:  Discrete colonies may be examined.During examination ,plates should be tilted in various directions under bright, direct examination so that is reflected from various angles.  Blood agar plates should be examined to when trans illuminated by bright light from behind the plate to the hemolytic reactions in the agar. The following are some of macro morphology features of the microorganism’s colonies: (a) Appearance-eg fluffy ,dry chalky, shiny etc (a) Size –diameters in millimeters is measured, small, medium or large colonies (b) Form-circular, filamentous, rhizoid, spindle like. (c) Elevation-flat ,raised, convex etc (d) Margin-Edge of the colony (e) Colour -white, yellow ,black ,buffy, orange, pink etc (f) Smell-fruity, feacal, sperm like smell, pungent ,fishy ,grape juice ,urine etc (g) Consistency-butter like, dry and granular ,mucoid (slimy dense growth) etc EXPERIMENT 3.3 Cultivation and isolation of fungi Fungi are the group of microorganisms which also causes various diseases in human and animals; Other fungi such as yeasts are beneficial to human being since they can be used in breweries industries, the need to isolate them is essential. Objective of the experiment To understand how to culture fungi (moulds and yeasts). Materials Sample: Dried fishes, groundnuts and vaginal swabs. Media: Sabourad´s dextrose agar (SDA). PROCEDURES  Sterilize a wire loop on flame  Make three holes in the media if the moulds are suspected  Add sample on the holes  Sterilize the wire loop before next sample is cultured  Duplicate should be made and one set place at room temperature and other set in the incubator at 37°C  Leave the plate for about three days and above  If yeasts are suspected in samples, the samples should be streaked in the media as we do in bacteria: This should be read after overnight.  Macro-morphology The macro morphology is read by observing the growth pattern:  Granular  Filamentous  Powdered  Pigmentation at the top and bottom of the media plates: White, black, Yellowish, Green etc NOTE: Yeasts colonies are read as bacterial colonies  EXERCISES: 1.Observe the plates you cultured on previous cultures you made and report the findings basing on the macro- morphology of the colonies. 2. State whether your culture media was contaminated before streaking or not contaminated with reasons. 3.If in your culture plate there are growth of mixed colonies, what do you propose as a medical scientist/personnel.? EXPERIMENT 3.4 ISOLATION TECHNIQUES/SUB-CULTURING Is the transferring of microorganisms from the present growth source to a fresh one. AIM; To be able to identify different colonies in a mixed culture and manage to perform isolation of pure culture. Materials:  Culture containing mixed colonies of bacteria  Straight wire  Bunsen burner  Fresh media.  Procedures  A bacterial colony is selected, the one which grow fairly well.  Sterilize the straight wire on flame  Remove the lid of the plate of the mixed culture with left hand and place on the bench without exposing the inner sterile surface.  Touch the selected colony with the tip of wire and return the plate to the bench , Replace the lid. Take a new media on left hand and remove the lid.  Streak the colony on the media and then replace the lid.  In case of broth, flame the mouth of the tube and introduce the straight wire into the medium  Flame the mouth of the tube and replace the plug.  The sub culture may now be incubated.The mixed culture may be kept in the refrigerator until the subculture has grown.  RESULTS / INTERPRETATION. A typical plate will show the presence of confluent growth where the initial streak will have less dense growth and discrete colonies will be seen. Any colony not growing on the streak marks is regarded as a contaminant.Choose a well isolated colony and record its features.