Unit 7 Isolation and Cultivation of Microorganisms PDF

UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms ISOLATION AND CULTIVATION OF...

UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms ISOLATION AND CULTIVATION OF Inoculum MICROORGANISMS microbes that are introduced into a culture medium to initiate growth PRINCIPLES OF BACTERIAL CULTIVATION Grow and isolate all bacteria present in Culture a clinical specimen microbes that grow and multiply in or on Determine which of the bacteria that a culture medium grow are most likely causing infection and which are likely contaminants Types: Obtain sufficient growth of clinically relevant bacteria to allow identification, a. Pure (Axenic) Culture- Composed of characterization, and susceptibility only one species testing b. Mixed Culture- Composed of more than Pure Culture one species A culture which contains a single species of microorganism c. Stock Culture- Composed of several A population of cells arising from a species contained in a separate culture single cell medium---one specie per culture medium. Grown in a large volume of Cultivation broth and then divided into small freezer process of growing microorganisms in vials---lengthen the shelf life of culture by taking bacteria from the specimen to at least a year infection site (in vivo environment) by some means of specimen collection Agar and growing them in the artificial Sulfated polymer made up of D- environment of the laboratory (in vitro galactose, 3,6-anhydro-L-galactose, environment) and D-glucoronic acid and usually Increasing the population of derived from red algae microorganisms by providing their melt at 80°C-90°C (100°C) and solidify nutritional and physical requirements at 40°C-50°C Nutrients Note!!! Extracellular substances which provide 55°C-60°C the cell with materials for building cooling temperature for distribution of protoplasm and for energy generation culture medium into Petri plates 20-25 ml Culture Medium Amount of molten agar transferred to a Any nutrient material for growth and sterile plate cultivation of microorganisms in the laboratory Uses of Culture Media composed of mixture of nutrients: For growth and maintenance of Carbon, Nitrogen, Sulfur, Phosphorus, microbial culture Hydrogen, Oxygen and Buffer To favor the production of particular Inhibitory agents facilitate isolation of compounds desired organism while suppressing the To study microbial action on some growth of other organism. constituents of the medium.  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms CLASSIFICATION OF CULTURE MEDIA According to Physical State or Consistency Examples: Triple Sugar Iron (TSI) Agar, MacConkey (MAC) Agar, Blood Agar Plate A. Liquid Medium (BAP), Chocolate Agar Plate (CAP) Commonly called broth in the microbiology laboratory According to Composition Media does not contain agar, but nutrients in the liquid A. Synthetic or Defined Medium The enrichment properties of the media All components are chemically defined enable microorganisms to be recovered exact chemical composition of the in higher numbers ingredients is known Is used for detecting the presence of a (commercially prepared culture media) low number of microorganisms that used for research purposes as either a may be missed if plated on solid agar liquid or solid medium only preferred for the isolation of Has specialized formulas that may be cyanobacteria and chemoorganotrophs used for biochemical testing and other Example: BG-11 medium uses allows growth of aerobes, anaerobes, B. Non-synthetic or Complex Medium and facultative anaerobes. Not all components are chemically defined Examples:Nutrient Broth,Brain Heart Infusion precise composition of some or all of (BHI),Trypticase Soy Broth (TSB), the nutritive substances used is not Thioglycollate (THIO) known (Peptone, Meat and Yeasts Extracts) B. Semi-solid Medium useful for the isolation of medically significant bacteria contains 0.5% to 1% agar Examples: Nutrient Broth (NB) Medium, TSB, Has the consistency between a liquid and MAC Agar potato infusion (of plant origin) and a solid beef extract (of animal origin) yeast extract (of Is often equated to jelly consistency microbial origin) Has a lower concentration of agar than solid media C. Tissue Culture Medium Is commonly used in determining contains living tissues organism motility used for obligate intracellular bacteria-- observed bacterial motility and detect -Rickettsia and Chlamydia indole and sulfide production Example: Sulfide Indole Motility (SIM) Medium Examples: McCoy Cells- mouse cell line C. Solid Medium Vero cells – African green monkey cell contains 1.5% to 2% agar A549- Lung Carcinoma A solidifying agent, such as agar, is HeLa Cells- Cervical Carcinoma added in the preparation of solid culture Hep 2 Cells- Laryngeal carcinoma media Does not inhibit bacteria or liquefy at room temperature Is used for developing surface colony growth of microorganisms Is critical for obtaining isolated colonies  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms According to the Dispensing or Distribution contain specific nutrients and without Method for the Medium additional supplements incubated for a certain period and then A. Plated Media subcultured to isolate the desired distributed into sterile petri dish organism can also be used as a supplement to B.Tubed Media agar plates to detect aerobes, distributed in sterile test tube Types: anaerobes and microaerophiles 1. Slant (THIOGLYCOLLATE) 2. Butt-Slant media with additional supplements 3.Butt necessary for the growth of fastidious Examples: TSI, SIM, Simmon’s Citrate organisms Agar (SCA), Lysine Iron Agar (LIA) Supplements: Blood, Vitamins, Serum, Peptone and Yeast Extract solid type media Example: Alkaline Peptone Water (APW) promotes growth of Vibrio spp. before inoculation into Thiosulfate-Citrate- Bile-Salts (TCBS) Agar; adjusted to pH 8.5 Selenite F isolation of Salmonella from feces, urine and water sample According to use Thioglycolate A. SIMPLE MEDIA, SUPPORTIVE MEDIA OR general support enrichment medium GENERAL PURPOSE MEDIA that promotes the growth of almost all routinely used in the laboratory and non-fastidious bacteria. without additional supplements support growth of most non-fastidious Components: bacteria to grow at natural rates, Dextrose, Vitamin K1, and Hemin have without providing advantage to any been used to modify the basic particular bacteria thioglycollate formula usually composed of meat and soybean 0.075% agar extracts Resazurin oxidation-reduction indicator Example: Nutrient Agar, Nutrient Broth, TSB, Thioglycolic Acid reducing agent Potato Dextrose Agar Tetrathionate B.ENRICHMENT MEDIA Selective enrichment broth for the used to increase the number of isolation of microorganisms with unusual Salmonella and Proteus physiological characteristics Bile Salt and Thiosulfate suppresses enhance the growth of particular the growth of other coliform bacilli organisms (pathogens) and suppress Gram-Negative Broth the growth of normal flora present in the isolation of Salmonella and Shigella specimen Enrichment and Selective medium  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Sodium Citrate and Sodium MacConkey Agar Desoxycholate (a bile salt) inhibit gram- differentiate Lactose Fermenter (pink positive organisms colonies) from Non-Lactose Fermenter (colorless colonies) Gram-Negative Broth Components: Lactose, Bile Salts, isolation of Salmonella and Shigella Crystal Violet inhibit gram-positive bacteria and fungi Enrichment and Selective medium Neutral Red: pH indicator Sodium Citrate and Sodium Desoxycholate (a bile salt)inhibit gram- D. SELECTIVE MEDIA positive organisms Mannitol primary carbon source support the growth of one type or group of microbes but not another Lim Broth (Todd Hewitt with CNA) contain inhibitory substances such as Group B Streptococci antimicrobials, dyes, or alcohol which inhibit the growth of other organisms Blood Agar Plate (BAP) while promoting the growth of the contains 5% defibrinated blood desired organism. Differentiate haemolytic pattern of bacteria Choices of blood: Sheep, Horse, Rabbit INHIBITORY AGENTS 1. Inhibit growth of Gram-Positive Microorganism Crystal or Gentian Violet Basic or Carbol Fuchsin Bile Salts Sodium Desoxycholate Chocolate Agar Plate 2. Inhibit growth of Gram-Negative blood has been chemically treated or Microorganisms heat-treated (80°C) to lyse the RBC Potassium Tellurite isolation of fastidious microorganisms: Sodium Azide Neisseria gonorrhoeae and Haemophilus spp 3.Prevent Swarming of Proteus Alcohol Note!!!! Chloral Hydrate Hemin - “X” factor Hektoen Enteric (HE) Agar Nicotinamide Adenine Dinucleotide o Salmonella spp. and Shigella (NAD)- spp. “V” factor o bile salts and dyes (bromthymol blue and acid fuchsin) C. DIFFERENTIAL MEDIA o Bromthymol Blue:pH indicator Blood Agar Plate Hemolytic pattern of Streptococci Ferric Ammonium Citrate- H2S indicator Eosin Methylene Blue (EMB) Salmonella-black precipitate Lactose and Sucrose Eosin and Methylene Blue Hektoen Enteric Agar (HEA) Xylose-Lysine-Desoxycholate (XLD) Agar  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Shigella spp. and Salmonella spp. Cefoperazone, vancomycin, amphotericin Components: (CVA)agar o Lysine, Lactose, Xylose and Selective medium for isolation of Sucrose Campylobacter spp. o 0.25% Sodium Desoxycholate- inhibits gram-positive bacteria Cefsulodin-Irgasan-Novobiocin (CIN) agar Phenol Red-pH indicator Selective for Yersinia spp.; may be Ferric Ammonium Citrate H2S useful for isolation of Aeromonas spp. indicator Salmonella colonies are red with black Chromogenic agar (CROM) center It is routinely used to identify yeast, MRSA, and other organisms. MEDIA FOR GRAM-POSITIVE BACTERIA a. Columbia CNA with Blood Cystine-tellurite blood agar Isolation of C. diphtheriae three peptone sources and 5% defibrinated sheep blood EMJH medium and Fletcher medium Colistin (C) and Nalidixic Acid (NA) Isolation of Leptospira interrogans suppress the growth of most gram- negative organisms Feeley-Gorman medium Legionella spp. b.Phenylethyl Alcohol (PEA) Agar sheep blood agar supplemented with Granada medium phenylethyl alcohol to inhibit the growth S. agalactiae of gram-negative bacteria. Kelly’s medium OTHER SELECTIVE MEDIA Borrelia spp Bile esculin agar Group D Streptococci and Enterococci Loeffler’s medium Isolation and growth of Blood glucose cysteine medium Corynebacterium Francisella spp. Lowenstein-Jensen medium Bordet-Gengou agar/Potato-Blood-Glycerol Egg-based medium for M. tuberculosis agar Isolation of B. pertussis and B. MacConkey agar with Sorbitol parapertussis For the selection and differentiation of E.coli 0157:H7 Buffered charcoal-yeast extract Enrichment for Legionella spp.; McBride Medium supports the growth of Francisella and Listeria monocytogenes Nocardia Regan Lowe/Charcoal-Cephalexin agar Campy-blood agar Enrichment and selective medium Selective for Campylobacter spp. for the isolation of Bordetella pertussis. Cycloserine-cefoxitin fructose agar Schaedler agar Clostridium difficile Selective for Campylobacter and Helicobater spp.  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Shepard’s medium PREPARING CULTURE MEDIA Mycoplasma General Steps in Preparation of Culture Medium Selenite broth Enrichment and isolation of Salmonella 1. Determine needed volume. spp. 2. Determine the amount of each component of the medium needed. Tetrathionate broth 3. Adjust pH if necessary. Selective for Salmonella and Shigella 4. Dispense in appropriate containers. spp. Except S. typhi 5. Sterilize in autoclave Thiosulfate-Citrate-bile salts agar TUBE METHOD Selective and differential for Vibrio spp. 1. Weighing- weigh the different ingredients then place them in clean, Todd-Hewitt broth with antibiotics (LIM) dry containers Selection and enrichment of S. 2. Dissolving- add the exact amount of agalactiae in female genital specimens solvent to the ingredients and then dissolve by heating Tomato Juice agar 3. Titration- adjustment to the right pH: Lactobacillus acidophilus 7.2-7.4 4. Distribution-distribute in test tubes Tap water agar 5. Sterilization To differentiate aerobic Actinomycetes (Nocardia, Rhodococcus, PLATE METHOD Streptomyces, etc.) 1. Weighing- weigh the different Gentamicin Blood Agar ingredients then place them in clean, Streptococcus dry containers 2. Dissolving-add the exact amount of Bacitracin Chocolate Agar solvent to the ingredients and then Haemophilus dissolve by heating 3. Titration- adjustment to the right pH: Blood Agar Plate with Ampicillin 7.2-7.4 AEROMONAS 4. STERILIZATION 5. DISTRIBUTION- distribute it to the Bacitracin Blood Agar tubes H. influenzae SPECIMEN CONSIDERATION Cystine Trypticase Agar Sterile body fluids, pus, urine, and Neisseria spp. sputum inoculated directly into the selected media McCoy Specimens received on swabs C. trachomatis can be inoculated directly into the culture media Specimens that require direct or “bedside” inoculations: Blood, Genital specimens, Corneal Scrapings, Sterile Fluids like Synovial and Peritoneal Fluids and  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Nasopharyngeal Swabs for isolation of c. Butt Medium Bordetella pertussis. Just stab the medium with an inoculating needle INOCULATION TECHNIQUES Streaking is the most common manner d. Butt/Slant Medium of inoculation Inoculate the butt first by stabbing the Specimen collected through a swab: needle to the bottom of the medium and gently roll the tip of the swab onto the then streak the surface in a zig-zag upper portion of the plate; inoculated manner toward the mouth of the tube. area should be streaked by a sterile loop afterward II. Inoculation of Plated Media Placement of fluid specimen or swabs a. Streak Plate Technique into a broth or liquid culture media isolate organisms in pure culture Stabbing of medium is usually performed with group A streptococci to b.Pour Plate create anaerobiosis and promote sub- determine the approximate number of surface hemolysis viable organisms in a liquid such as Overlapping inoculation used for water, milk, urine, or broth culture antimicrobial sensitivity test c.Streak-Pour Plate for studying hemolysis. I. Inoculation of Tubed Media a. Liquid Medium with the use of sterile Pasteur pipet inoculate by shaking a previously heated wire loop or needle b. Slant Medium With the use of a wire loop or needle, transfer the inoculum to the bottom of the slant and streak in a zig-zag manner across the entire surface toward the tube’s mouth.  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Streak Plating QUANTITATIVE ISOLATION Urine specimens use a calibrated loop to deliver a specified volume: 0.01 or 0.001 Ml A calibrated loop inserted into the urine and transferred to the culture medium by making a single streak down the center of the plate without flaming, the loop is streaked back and forth through the original inoculum. Manner of Reporting (Grading) of Growth on Plate 4+ = many, heavy growth; growth is up to the fourth quadrant 3+ = moderate growth; growth is up to the third quadrant 2+ = few or light growth; growth is in the second quadrant 1+ = rare growth; growth is in the first quadrant only  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms includes form, elevation, and margin of METHODS OF OBTAINING PURE CULTURE the colony Streak-plate method Colony surface appearance Pour- plate method glistening, opaque, dull, dry, Use of selective media and media transparent containing antibiotic Animal inoculation test Changes in agar media resulting from bacterial growth COLONY MORPHOLOGY hemolytic pattern on blood agar, Colony size changes in color of pH indicators, usually measured in millimeters or pitting of the agar surface described as pinpoint, small, medium, large Odor certain bacteria produce distinct odors Colony pigmentation that can be helpful in preliminary Colony shape identification TYPES OF COLONY A. Mucoid (M) Colony exhibits a water-like, glistening, confluent appearance characteristic of organisms that form slimes or well-developed capsules Examples: K. pneumoniae, S. pneumoniae, H. influenzae  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Culture Media: Blood Agar (BAP) B.Smooth (S) Colony uniform texture and homogeneity TYPES OF HEMOLYSIS easily emulsified in NSS A. Alpha Hemolysis/α-Hemolysis characteristic of freshly isolated wild type microorganisms (virulent partial lysing of erythrocytes in a BAP microorganisms) around and under the colony that Examples: Salmonella, Shigella, E. coli, results in a green discoloration of the Serratia, Proteus medium Example: Streptococcus pneumoniae,and certain viridans streptococci) CHECK LETTER IN THE PICTURE : ALPHA HEMOLYSIS B B. BETA HEMOLYIS/β-Hemolysis C.Rough (R) colony complete clearing of erythrocytes in BAP around or under the colonies granulated and rough in appearance because of the complete lysis of RBCs hard to emulsify in NSS usually produced by mutant strains that Examples: lack surface proteins or Streptococcus pyogenes wide, deep, polysaccharides indicating loss of clear zone of β- hemolysis, virulence Streptococcus agalactiae and Examples: Rough forms of enteric bacteria Listeria monocytogenes Exception: R forms of B. anthracis and human narrow, diffuse zone of β- hemolysis and bovine types of M. tuberculosis (more close to the colony virulent) Select Letter A for Beta Hemolysis Gross Colony Characteristics Used to Differentiate and Identify Microorganisms Presumptively A. Hemolysis observed in the media immediately surrounding or underneath the colony is a reaction caused by enzymatic or toxin activity of bacteria presumptive identification of streptococci and enterococci  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms C. Gamma Hemolyis No Clearing in the BAP at all due to absence of enzyme to hemolyse the blood in the agar. Example: Enterococcus faecalis (formerly called "Group D Strep"), Staphylococcus saprophyticus, and Staphylococcus C. Form or margin epidermidis display gamma hemolysis. edge of the colonies Select Letter C for Beta Hemolysis described as smooth, filamentous, rough or rhizoid, or irregular Examples: Bacillus anthracis “Medusa Heads”--- filamentous appearance Proteus mirabilis and Proteus vulgaris swarming MOTILITY phenomena Swarming hazy blanket of growth on the surface that extends well beyond the streak lines Diphtheroids have rough edges D. Double Zone Hemolysis/ target hemolysis Clostridium perfingens - is a readily identified in the lab by its characteristics “double zone” hemolysis also known as TARGET HEMOLYSIS E. Alpha prime (a’)/ wide zone hemolysis S. vividans- small area of infected RBC’s around colony surrounded a widen zone D. Elevation of complete hemolysis Some isolates belonging to the vividans determined by tilting the culture plate group produce what is called as wide- and looking at the side of the colony zone or alpha prime hemolysis raised, convex, flat, umbilicate (depressed center, concave—an B. Size “innie”), or umbonate (raised or bulging described as large, medium, small, or center, convex—an “outie”) pinpoint gram-positive bacteria produce smaller Examples: colonies than gram-negative bacteria S. Pneumoniae umbilicate colonies Staphylococcus spp. are usually larger (unless the colonies are mucoid) than Streptococcus spp. S. aureus convex colonies β-hemolytic streptococci flat colonies determined by tilting the culture plate and looking at the side of the colony  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms raised, convex, flat, umbilicate (depressed center, concave—an “innie”), or umbonate (raised or bulging center, convex—an “outie”) Examples: F. Color S. Pneumoniae umbilicate colonies white, gray, yellow, or buff (unless the colonies are mucoid) S. aureus convex colonies Example: β-hemolytic streptococci flat colonies Coagulase-negative staphylococci white Enterococcus spp. Gray Certain Micrococcus spp. and Neisseria (nonpathogenic) spp. yellow or off-white Diphtheroids buff Most gram-negative -gray on BAP G. Consistency determined by touching the colony with a sterile loop brittle (splinters), creamy (butyrous), dry, or waxy Example: S. aureus creamy Nocardia spp.brittle, crumbly, and wrinkled, resembling breadcrumbs on a plate Diphtheroid dry and waxy E. Density transparent, translucent, or opaque Examples: β-Hemolytic streptococci except group B (S. agalactiae)- translucent S. Agalactiae- semi opaque Staphylococci and other gram-positive bacteria opaque Most gram-negative rods are opaque Bordetella pertussis shiny, like a half- pearl, on blood-containing media  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms H. Pigment Pseudomonas aeruginosa green Serratia marcescens brick-red (especially at room temperature) Chromobacterium violaceum purple Prevotella melaninogenica brown black (anaerobic) Kluyvera spp.blue Colonial Morphology  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Colonial Morphology I.Odor S. aureus old sock---Mannitol Salt Agar P. aeruginosa fruity or grapelike Proteus mirabilis putrid Haemophilus spp. musty basement, “mousy” or “mouse nest” smell Nocardia spp.freshly plowed field  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms Ways to facilitate Anaerobic Cultivation Decolorization of indicator strip: a. Special culture medium incorporated WHITE or COLORLESS with Thioglycollate and Cystein (reducing agents) B. Glove Box or Anaerobic Chamber b. Boiling of culture medium c. Anaerobic chamber system with a Enclosed system consists of large clear vacuum pump and nitrogen gas to plastic, airtight bag or chamber filled remove residual oxygen with oxygen-free gas mixture of d. Gas-pak jar containing a palladium nitrogen, hydrogen, and CO2 catalyst Allow materials to enter through an air e. Small volumes: plastic bags, pouches lock containing calcium carbonate and Anaerobiosis---maintained by catalyst Palladium catalyst and Hydrogen gas Operator uses gloves or sleeves that form airtight seals around arms to manipulate items ANAEROBIC CULTIVATION ANAEROBIC CULTURE METHODS A.Anarobe Jars 1.Brewer Jar Oxygen is removed by means of electrically heated platinized catalyst with the electrical connection outside the jar 2.Torbal Jar uses a rubber O ring rather than Plasticine and a catalyst active at room Components temperature thus requiring no electrical heating a.Nitrogen Gas filler for the remaining percentage of the 3.GasPak Jar anaerobic structure most convenient and widely used b.Palladium Pellets anaerobic jar remove residual oxygen by combining takes 30 to 45 minutes to obtain with H2 to form water anaerobic environment c.silica Gel (Desiccant) Principle: absorbed water With water added to the CO2 and H2 d.Methylene Blue or Resazurin generator envelop and oxygen catalyzed oxygen reduction indicator with H2 to water via the pellets, anaerobiosis is achieved Indication of Anaerobiosis Production of heat Moisture inside jar  UNIT SEVEN ISOLATION isolation AND and CULTIVATION cultivationOF of MICROORGANISMS microorganisms C. Role Tube Technique Pre-Reduced Anaerobically Sterilized (PRAS) agar is distributed under anaerobic conditions as thin layer around the inner wall of test tubes tubes are rolled and cooled until the melted agar forms the thin layer Inoculated in 2 ways: Closed Method by Hungate -Syringe and needle are used through the rubber seal Open Method-remove the rubber stopper and insert a cannula that has oxygen-free gas flowing from the tip.