Diagnostic Microbiology Lab PDF

Lab NO. 1 Diagnostic Microbiology Lab Introduction and Safety in the laboratory INTRODUCTION Microbiological techniques are different in many ways when compared with other laboratory disciplines. Although results are not obtained in a s...

Lab NO. 1 Diagnostic Microbiology Lab Introduction and Safety in the laboratory INTRODUCTION Microbiological techniques are different in many ways when compared with other laboratory disciplines. Although results are not obtained in a short time, the time required to perform the test is very short. Most of the techniques are simple, yet requires a great deal of theoretical background to be correctly interpreted. Before performing any of the exercises in this manual, one should read the safety precaution and measures as well as the exercise (the materials needed, the procedures, and the expected results). This will ensure the safety of the student and also will ensure good results. General View on the Parameters Used in the Process of Microorganism Identification Before one can proceed to identify a microorganism, the characteristics of that organism have to be determined in details. The major characteristics which are observed include the following: A. Cultural Characteristics In clinical terms, it is the shape, size, color, elevation and other characteristics of the colony formed on the culture plate. In taxonomy, it includes the nutrient requirements for the growth of the organism and the physical factors such as temperature, pH and the incubation period. These factors are used to identify certain pathogenic species but less commonly used in routine procedures. The cultural characteristics of a microorganism usually vary depending on the media used and many other factors. Some experienced microbiologists could have a good guess about the identity of a microorganism just by its cultural characteristics, but this was proven to be a bad technique. Students as well as microbiologists are advised to follow strict procedures for the identification of isolates from clinical specimens. B. Morphology and Staining This includes the microscopic appearance of a stained preparation of the organism. Useful information to be taken into account, are the size of the individual cells, cell shape and arrangement and staining reaction if differential staining procedures is used. EXAMPLE: A gram stained film prepared from a pure culture of certain microorganism shows the following: -Small spherical cells "Cocci" -Arranged in clusters -Gram-positive = violet in color Some laboratories which have a little facility could give the report of a microbiological examination of a clinical specimen just by stating their morphological characteristics and the sensitivity testing results. C. BIOCHEMICAL CHARACTERISTICS Frequently, the identity of a species requires detailed knowledge of its biochemical activities, since other characteristics are not sufficiently distinctive or differential. For example, the bacterium Escherichia coli, a normal inhabitant of our intestinal tract, is indistinguishable microscopically from Salmonella typhi, the bacterium that causes typhoid fever. However, if these two bacteria are examined for their metabolic (or biochemical) characteristics, they are found to be very different and distinguishable on this basis. Numerous laboratory techniques are available for the characterization of microorganisms. D. SEROLOGICAL CHRACTERISTICS Sometimes, to identify a species as E. coli is insufficient, for the reason that some strains of this organism are non-pathogenic and others are highly associated with diseases. Serological testing in such case will identify the exact strain number based on testing against prepared specific antisera. General Information The microbiology laboratory is considered to be vital and the take the great amount of the general work load of the laboratory. Receiving and recording specimens, culturing, staining, isolation and identification of pathogens and doing sensitivity tests for the isolated pathogens are the major tasks. Information For Microbiology Laboratory Staff General Requirements for Collecting and Submitting Specimens Proper collection and adequate amounts of specimen are required. The following criteria should be used as guidelines: Medical Group employees who handle laboratory specimens have relatively high rates of work-related hepatitis and other transmittable diseases. Loosely capped containers and soiled requisitions sent to the laboratory are a significant risk to all who come in direct contact with these contaminated materials or areas contaminated by such materials. Therefore, laboratory staff will not accept soiled laboratory requisitions/leaking specimen containers. When needed, a written test request must include the following information: Patient details 􀂉 Hospital No. 􀂉 Name: First name and family name 􀂉 Sex 􀂉 Date of birth/Age 􀂉 Address 􀂉 For females: whether pregnant or lactating 􀂉 Details of illness 􀂉 Presenting signs/symptoms 􀂉 Duration/date of onset 􀂉 Recent travel history 􀂉 Immunizations Identification of Specimens 􀂉 Type of specimen (Exact source and nature of specimen) 􀂉 Collection date and time 􀂉 Tests requested 􀂉 Ordering physician Also, ALL specimens must be properly labeled with the patient's full name, date and time of collection, and specimen source. Swab Specimens: Separate specimens must be submitted for each specific request, i.e., one for bacterial culture, one for fungal culture, etc. Special Culture / Specimen Requirements: Anaerobic Specimens: Submit in anaerobic transport containers or in a sealed syringe with no bubbles. SPECIMEN COLLECTION Proper specimen collection, container labeling, and culture requests are the responsibility of the ordering physician. Technologists in the Clinical Microbiology Laboratory will be familiar with specimens of choice and proper collection techniques. The technologist in the laboratory will directly handle specimens of clinical and environmental source which are received from the Postal Service or hand carried to the laboratory. SPECIMEN DELIVERY The Clinical microbiology Laboratory recommends that a specimen should be transported to the laboratory as soon as possible (a maximum delay is indicated for each type of specimen. If more than one specimen is received for one type of analysis, the Processing Area staff will note on the requisition "Duplicate Specimen" in red ink. Actively growing cultures of organisms for identification should be submitted on tubed media appropriate for the organism being submitted. Seal the tube with water proof tape. All specimen containers should be closed tightly or sealed in order to prevent leakage and contamination. Media in Petri dishes or liquid cultures are not an acceptable transport media. SPECIMEN REJECTION CRITERIA. General Issues In general, specimens for the microbiology laboratory are unacceptable if any of the following conditions apply: 1. The information on the label doesn’t match the information on the request form. 2. The specimen was transported in an improper container or at wrong temperature. 3. The quantity of the specimen is insufficient to carry out all the required examination. 4. Leaking specimen Lab NO. 2  Staphylococcus and Micrococcus Gram positive Cocci: 1- Micrococcaceae: - Micrococcus sp. - Staphylococcus sp. 2- Deinococcaceae: - Streptococcus sp. Procedure of biochemical test used in the identification of Staphylococcus species: Catalase test: Procedure: Slide method: recommended procedure. 1- With an inoculating loop, take large inoculums of pure colony and place it on a clean glass slide. 2- Add one drop of 3%H2O2 directly over the organism on slide. Use a dropper or Pasteur pipette. 3- Observe for immediate bubbling (gas liberation) and record result. 4- Discard slide. Coagulase test: Procedure/Slide method: 1- Place one drop of plasma at one end of a clean slide. 2- At the second end of the slide add one drop of distilled water then transfer large amount of bacterial growth into distilled water drop and mix it well to bring homogenous suspension. 3- Mix the two drops well. 4- Result should be taken with 10-15 seconds. Positive: white clumping Negative: homogenous suspension with no white clumping (this result should be confirmed by tube method) Tube method: 1- Dispense 0.5 ml of sterile plasma into clean tube. 2- Add 0.5 ml of bacterial broth or two loopful from solid culture. 3- Mix the tube between your hands. 4- incubate the test tube at 35 C for 4 hours. 5- Result should be taken every 30 minutes. If the result remains negative reincubate the tube for over night. 6- Result: Positive: fibrin clot formation Negative: no fibrin clot Novobiocin resistant test: Procedure: 1- With sterile cotton swab, transfer one colony to inoculate blood agar plate. 2- Move the swab by three directions horizontal, vertical and around the edge of plate to ensure equal distribution. 3- With sterile forceps apply the disc of Novobiocin to the inoculated plate. 4- Incubate the plate at 35C for 24 hours. 5- After incubation period with ruler measure the zone of inhibition. 6- If the zone of inhibition is more than 16mm in diameter the result will be negative(Sensitive), if the zone of inhibition is less than 16mm in diameter the result will be positive ( Resistant). Oxidation fermentation test: O-F basal media (semi solid) compose of: -Glucose as carbohydrate - Bromothymol blue ad indicator - Other ingredient Procedure: 1- Inoculate two tubes with an organisms (stabbing). 2- Add to one of them 6 drops of sterile mineral oil to create anaerobic condition. 3- Incubate both tubes in the incubator at 35C for 24 hours. 4- After incubation period, take the result. - If the color of both tubes change to yellow; Oxidation and Fermentation - If the color changed to yellow only in covered tube (mineral oil) and not changed in other tube (remain green): Fermentation, and no oxidation - If the color not changed in the covered tube (remain green) and changed to yellow in the other tube: Oxidation and no fermentation. - If the color not changed in both of them (remain green): No oxidationand no fermentation. Gram positive cocci/ Micrococcus sp. Catalase positive Non motile Non spore forming Aerobic organisms Anaerobic glucose fermentation Old cells may lose their ability to retain crystal violet in gram technique. Morphological shape of bacterial cells: Spherical with 0.5 - 2.0Mm in diameter, occurring mostly in pairs tetrads or irregular clusters. Culture characteristic: Colonies appear circular, entire, convex and smooth. Colonies may have a yellow, orange, orange-red or red pigment. Gram positive cocci /Staphylococcus sp. -Catalase Positive - Non motile - None spore forming -Aerobic ad facultative anaerobic organisms -Anaerobic glucose fermentation: (positive) - Old cells may lose their ability to retain crystal violet in Gram stain technique. Culture characteristic: Spherical with 0.5-1.5 um in diameter, occurring singly, pairs, irregular clusters and short chains are also seen. Morphological shape of cells: Colonies appear, circular, raised convex, opaque and smooth. Colonies may have white pigment gray, creamy, golden yellow pigment. Gram positive cocci /Staphylococcus aureus Coagulase test: positive Novobiocin resistant: negative (sensitive ) Mannitol fermentation: positive (Fermenter) Morphological shape of cells:single, in pairs or clusters. Culture characteristic: - Colonies appear on blood agar media, medium to large with Beta hemolytic (most of them). It has golden pigment produced at room temperature or at over incubation period (several days). Gram positive cocci/Staphylococcus epidermidis Coagulase test: negative Novobiocin resistant: negative (sensitive) Mannitol fermentation: negative (non fermenter) Morphological shape of cells: Single, in pairs or clusters Culture characteristic: Colonies appear small to medium snow-white or gray colonies. Without any hemolytic (some time give weak reaction of beta hemolytic). Gram positive cocci /Staphylococcus saprophyticus Coagulase test: negative Novobiocin resistant: positive (resistant) Mannitol fermentation: positive (fermenter) Morphological shape of cells: Single, in pairs or clusters. Culture characteristic: - Colonies appear large smooth, opaque, convex colonies with white oryellow orange pigment. Lab NO. 3  Streptococcus sp. Optochin Susceptibility Test: Procedure -Streak one quadrant or one half of a sheep blood agar or chocolate agar plate with an inoculum of the organism to be tested (pure isolate). - Place an Optochin disk on the center of the inoculum using sterile forceps Incubate the plate overnight (24 hours) at 35 °C in the present of 5-10% of CO2 or in candle jar. - After incubation period, observe the zone of inhibition surrounding the disk. Zones of equal or greater than 14 mm surrounding a 6 mm diameter disk and zones equal to or greater than 16 mm surrounding a 10 mm diameter disk are considered sensitive S. pneumonia. Zone size of inhibition between 6-10mm (6 mm disk) and 10-16mm (10mm disk) are equivocal result, and those isolates should be tested for bile solubility. Bile solubility test: Plate method: - Select well isolated colony from blood or chocolate agar plate and place one drop of the reagent (10% sodium Deoxycholate) directly upon the colony. Keeping the plate very level, to prevent the reagent from running and washing a non-pneumococcal colony away, producing a false positive result. Placing the plate in an aerobic incubator will speed up this process (35°C for 30 minutes). - When the reagent has dried examine the area for the absence (dissolves) of the original colony -Absence or dissolves of the original colony Positive result. Tube method: - Inoculate 5ml tube of serum with tested an organism. - Incubate the test tube at 37°C for 18 hours. - After incubation period while the tube still warm add 0.5 ml of 10% sodium Deoxycholate and reincubate at 37 °C for 30 minutes. - Bile soluble culture will appear clear (positive result) - Bile insoluble culture will be turbid ( negative result) Bacitracin Susceptibility Test: 1. Streak one half of a sheep blood agar or chocolate agar plate with an inoculum from a pure isolate of the organism to be tested 2. Place a 0.04 U Bacitracin disk on the center of the inoculum using sterile forceps. 3. Incubate the plate overnight at 35 °C in 5% - 10% of CO2 or in candle jar. 4- After incubation period, observe for zone of inhibition surrounding the disk. Any inhibition of zone is indicative of Bacitracin susceptibility Sodium Hippurate Hydrolysis Test: - Inoculate a plastic disposable test tube that contains 0.4ml of 1% sodium hippurate with heavy inoculum (pure culture). The suspension should be milky. - Incubate the capped tubes for 2½ hours in a 35 °C water bath. - After incubation period, add 0.2 ml ninhydrin reagent and re- incubatefor additional 15 minutes. 3. Observe for a deep blue color change, indicating that the hippurate has been hydrolyzed (positive). The color change will usually appear in 10 to 15 minutes after the Ninhydrin Indicator Solution has been added. A negative reaction is indicated by a faint blue color change, or no color change.  CAMP Test: - Inoculate a streak line of Staphylococcus aureus down the center of a 5% sheep blood agar plate. - Inoculate straight line of the isolate organism to be tested at right angle to the S. aureus streak stopping just before the S. aureus line is reached. - Incubate the plate-at 35-37 °C overnight in air or for 6 hours 5-10% CO2. - After incubation period, observe for an arrowhead shaped zone ofenhanced Hemolysis at the juncture between Streptococcus and S aureus. Bile Esculin Test: - Inoculate one or two colonies from an 18-24 hours culture onto the surface of slant tube (fish tail) or on the surface of plate that contains bile esculin media. - Incubate the test tube or plate at 35 °C for 18 up to 72 hours (3 days) - Positive result : present of black to dark brown color (halve or more of the medium is blackened) - Negative result: No blacking of the media. NaCI %6.5 : - Using a sterile loop transfer the organism to be tested to the 6.5% NaCI broth. - Incubate the tube for a minimum of 18 hours. - Examine the tube for evidence of growth (turbidity). It may be helpful to compare the tube to an uninoculated tube Do not agitate the tubes before you examine them. - Interpretation: Organisms that can tolerate a high salt environment (6.5% NaCl) will grow in this broth causing the broth to become cloudy or turbid. Turbidity is considered positive(+) for this-test..Clear broth is considered negative (-) for this test. Deinococcaceae Family: Streptococcus sp.: Catalase negative Non motile None spore forming Aerobic or facultative anaerobic organisms Need 5-10 % of Co2 Morphological shape of bacterial cells: - Gram positive Cocci (round or oval shaped). Streptococcus species may appear Gram negative if culture are old or if there has been treatment with an antibiotic. Cells appear Spherical less than 2 um in diameter occurring single in pairs or chains when grown in liquid or broth media. Culture characteristic (colony description) -Pinpoint or Small to medium. - Most species are none pigmented except some strain of S. agalactia and some Enterococeus which may have yellow, orange or red coloredcolonies. Streptococcus pneumoniae: Cells: Cocci shape in pairs or short chains, lancet shape (diplococcus). Colonies: Small, round, gray, glistening and some times mucoid due to capsule. All colonies surrounding with alpha hemolytic (greenish zone). Streptococcus pyogenes (Beta hemolytic group A): - Cells: Cocci shape, single, and mostly in chains (broth culture). - Colonies: Circular, shiny, transparent to translucent, pin point (verysmall) colonies surrounding with wide zone of beta hemolytic. Streptococcus agalactia (beta hemolytic group B): Cells: Cocci shape, single or in pairs. Colonies: Larger than group A, translucent to opaque, flat, surroundingwith narrow zone of beta hemolytic (some strain non hemolytic). Group D Enterococci:Streptococcus faecalis: May grow in MacConkey agar mostly of these organisms are none hemolytic (gamma hemolytic) but some times may be give alpha or beta hemolytic. Colonies appear small, creamy- white, smooth, and entire. Lab NO. 4 Procedure of Biochemical Tests 1. Motility test: Motility media are SIM media and MIO media. 2. IMVIC Reaction: Stands for: ▪ Indole test ▪ Methyl red test ▪ voges-Proskauer test ▪ Citrate test INDOLE TEST Principle: To test the ability of an organisms to split indole from the Tryptophane molecule. (Media: Tryptophane broth or peptone broth, or SIM media or MI0, Reagent: Kovac's) Procedure: 1. Inoculate the test tubes f Tryptophane broth with a loopful of bacterial growth. 2. Incubate the tubes at 35-37° C for 24 hours. 3. After incubation period, open the test tube add 5 drops of Kovac’s reagent. 4. Read the result immediately Positive----- Pink or red ring. Negative --- yellow ring. METHYL RED TEST Principle: To determine the ability of an organisms to produce acid as final products from glucose fermentation. (Media: MR/VP broth, Reagent: Methyl red Procedure: Inoculate the test tubes of MR/VP broth with a loopful of bacterialgrowth. 1. Incubate the tubes at 35-37 °C for 48 hours. 2. After incubation period, add 4 drops of Methyl red reagent. 3. Read the result immediately Positive --- Red Negative --- Yellow VOGES PROSKAUER TEST Principle: To determine the ability of an organisms to produce acetoin as final products from glucose fermentation. (Media: MR/VP broth, Reagent: 1- Alpha naphthol, 2- 40% KOH) Procedure: 1. Inoculate the test tubes of MR/VP broth with a loopful of bacterialgrowth. 2. Incubate the tubes at 35-37C for 48 hours. 3. After-incubation period, add to the tube ( 1ml of bacterial broth), 6drops of Alpha naphthol and 2 drops of 40% KOH reagents. 4. Loosely the cap of tube and place it in slant position for 15 to 45 minutes. 5. After 15 minutes read the result if it is positive report it and if it is negative keep it for another 30 minutes. Red --- Positive Yellow --- Negative CITRATE TEST Principle: To determine if the organism is capable to utilize citrate as the only sole source of carbon. (Media: Simmon's Citrate agar, Indicator: Bromothymol blue). Procedure: 1. Inoculate the slant of test tubes with an organism as fish-tail. 2. Incubate the tubes for 1 to 4 days at 35-37 °C with loosely cap 3. After incubation period read the result immediately. Deep blue ----- Positive No Change (Green) ----Negative KLIGLER IRON AGAR (KIA) Principle: To determine the ability of microorganism to ferment carbohydrate (glucose & lactose), determine gas production (CO2) andHydrogen sulfide gas production. Formula: Glucose 0.1% Lactose 1% peptone Phenol red Sodium Thiosultefate Ferric Ammonium Citrate (Media: KIA, Indicator Phenol Red) H2S (colorless gas) reacts with IRON in the medium (ferric ammonium sulfate as H2S indicator) to produce a BLACK PRECIPITATE (ferrous sulfide FeS. H2S+ ferrous sulfate or ferric ammonium citrate ferrous sulfide Procedure: 1. Inoculate the test tube of KIA with an organism: Stabbing in the butt then Fish tail on the slant. 2. Incubate the tubes at 35-37°C for 24 hours (Time is Critical). 3. Read the result immediately after the incubation period. UREASE TEST Principle: To determine the presence of Urease enzyme. (Media: Urea broth media or Urea agar/ slant, Indicator Phenol Red). Procedure: 1.The surface of slant agar media is streaked with the test organisms asfish-tail (Urea agar/ slant) 2. Incubate the tubes at 35-37°C for 24 up to 72 hours. 3. After incubation period read the result: Red to pink color --- Positive No change (yellow) --- Negative OXIDASE TEST Principle: To determine the presence of Cytochrome oxidase enzyme Procedure: pick up one or two isolated colonies by a wooden applicator stick and rub it directly on filter paper impregnated with N. N. N. N. Tetramethyl paraphenylen diamine dihydrochloride (TPD) as reagent. Read the result within 10 second (Do not use wire loop or needle cause a false positiveresult). Dark purple or blue color --- Positive No change --- Negative Enterobacteriaceae This family divided into two groups according to their ability to ferment lactose: 1. Lactose fermenter groups. »Escherichia coli »Klebsiella sp. »Enterobacter sp. »Citrobacter sp. 2. None lactose fermenter groups. »Proteus sp Salmonella sp Shigella sp »Morganella Morgani »Providencia sp »Seratia sp »Yersinia pestis Characteristics of Enterobacteriaceae family: - Gram Negative slightly short or slightly longer rods, single - Some of genera are motile due to presence of peritrichous flagella and other genera are not motile. - Aerobic or facultative anaerobic. - All genera of this family ferment glucose. - Oxidase: Negative. - Reduce nitrate to nitrite. - Catalase: Positive. - Colonies: Large, gray and smooth with beta or gamma hemolysis onBAP and some genera give mucoid colonies. Identification of Enterobacteriaceae members: - Growth of organisms on Blood and Chocolate agar mostly Look the same. Gram stains... Look the same (Gram-Negative rods). Oxidase Test MacConkey Agar Kligler Iron Agar (KIA). Nitrate reduction test. IMVIC Reaction Motility Test Urease Test Identification of Enterobacteriaceae mnembers 1- E. coli -EPEC (Enteropathogenic ) -ETEC (Enterotoxigenic ) -EHEC (0157) H7 (Enterohemorrhagic ) -EIEC (Enteroinvasive ) -EAEC (Enteroaggregative) - Gram Negative bacilli single - Sometime give beta hemolysis in blood gar On MacConkey agar: E. coli can be, Lactose fermenter or non- Lactosefermenter Colonies appear smooth and large, On Eosin Methylene Blue agar media(EMB). It gives green metallic sheen colonies (greenish to silver color). IMVIC: + + - -- KIA: A/A,G A/A,G, H2S K/A,G, & K/A Motility: Motile Urease: Negative H2S: Mostly Negative 2- Enterobacter aerogenes: Gram-Negative Bacilli, single n MacConkey agar: colonies appear mucoid pink-red IMVIC: - - ++ KIA: A/A,G, KIA,G Motility: Motile Urease: Negative except Enterobacter cloacae H2S: Negative 3- Klebsiella sp Gram-Negative Bacilli, single On MacConkey agar: colonies appear large slimy viscous (gummy) or mucoid with pink-red color. IMVIC: - - + + (K. pneumonia e) + - + + (K. oxytoca) KIA: A/A,G Motility: Not Motile Urease: Positive H2S: Negative 4- Citrobacter sp Gram-Negative Bacilli, single On MacConkey agar: colonies appear pink-red mucoid. Ferment lactose weakly. IMViC: - + - + Citrobacter freundii  + + - + Citrobacter sp KIA: K/A, H2S, A/A,H2S Citrobacter freundii A/A,G Citrobacter sp Motility: Motile Urease: Positive Lab NO. 5  Gram Negative Bacilli Enterobacteriaceae None Lactose fermenter group 5- Proteus sp. - Gram Negative Bacilli - single On MacConkey agar: colonies appear colorless with foul smell. - On blood and chocolate agar:- it gives swarming (like the wave of the sea) To prevent swarming: - Cystine Lactose Electrolyte Deficlent (CLED). It Is used for differentiation of microorganisms in urine - Phenyl Ethyl Alcohol PEA - Duplicate The concentration of Agar in The Media IMVIC: ++-- P. vulgaris - + (-/+) (-/+) P. mirabilis KIA: K/A,G,H2S A/A,G,H2S Motility: Motile Urease: Positive H2S: Positive 6- Marganella morgana - Gram Negative Bacilli, single - It have all Proteus character except it doesn't produce H2S IMVIC: + + - - KIA: K/A,G Motility: Motile Urease: Positive H2S: Negative 7- Salmonella sp. Gram Negative Bacilli, single On MacConkey agar: colonies appear colorless On Salmonella Shigella agar medium, colonies appear colorless with black center (H2S). IMVIC: -+-- S. typhi& S. paratyphi A -+-+ Other type of Salmonella KIA: K/A,H2S S. typhi K/A, G S. paratyphi A K/A, G,H2S Other type of Salmonella Motility: Motile Urease: Negative H2S: Positive except S. paratyphi A (10%: +) 8- Shigella sp. Gram-Negative Bacilli, single On MacConkey agar: colonies appear colorless and flat. On Salmonella Shigella agar medium, colonies appear colorless. IMVIC:(-/+) + - - ( serogroups A,B,C) -+-- S. sonnei (group D) KIA: K/A Motility: Not Motile Urease: Negative H2S: Negative 9- Providencia sp. Gram Negative Bacilli, single -On MacConkey agar: colonies appear colorless Similar to Proteus (without swarming in BAM) IMVIC: KIA: K/A,G K/A Motility : Motile Urease: Negative except P. rettgeri H2S: Negative 10- Serratia marcescens Gram Negative Bacilli, single On MacConkey agar: colonies appear none lactose fermenter with Briek red pigment. On blood agar and nutrient agar media colonies appear with brick red pigment. IMVIC: KIA: K/A,G K/A Motility: Motile Urease: Negative H2S: Negative 11- Yersinia pestis Gram-Negative Bacilli, single On MacConkey agar: colonies appear colorless IMVIC: - + - - KIA: K/A Motility: Not Motile Urease: Negative H2S: Negative Salmonella Shigella agar medium SS agar is a selective medium for isolation of Salmonella and Shigella from stool and food Product. Inhibitor factors: Brilliant green Bile salt Sodium thiosulfate Sodium citrate PH indicator: Neutral red Source of Carbohydrate: Lactose Examine the media for non-fermenting colonies (colorless), with or without H2S. Colonies with black center confirm H2S production. - Wash with tap water. - Blot dry and examine under oil immersion lens. Cautions: Do not allow stain to evaporate; (add stain as needed). Prevent the stain from boiling Gelatin Liquefaction Test Purpose: This test is used to determine the ability of microorganism to produce hydrolytic exoenzyme called gelatinase that digest and liquefy gelatin. Procedure: 1- Inoculate the test tube of gelatin media with an organisms using stabbing technique. 2- Once the tubes are inoculated, they will be incubated at 37 °C for 24 hours. This will cause the gelatin to melt. 3- After incubation period, the incubated tubes must first be refrigerated until the control tube is once again solid (approximately for 10-15 minutes). When tubes are first taken out of the incubator, the gelatin will be liquid due to the warmth of the incubator (35-37°C). Result: Negative: (gelatin is solid) Escherichia coli Positive: (gelatin has been liquefied) Bacillus sp. , Clostridium sp.,Pseudomonas aeruginosa. Starch Hydrolysis Test Purpose: This test is used to differentiate bacteria based on their ability to hydrolyze starch using the exoenzyme amylase. Principle: Starch is a polysaccharide molecule which is too large to pass Through the bacterial cell membrane. These large molecules can be hydrolyzed into smaller fragments or individual glucose molecules by extra cellular enzymes amylase. The smaller molecules can then enter the cells. Procedure: - Inoculate the plate of starch media with an organism using streak plate method(Single Streak Line). - Incubate the test plate at 35 °C for 24 hours. - After incubation period add few drops of diluted iodine reagent directly over the plate. - After adding iodine The blue color indicates the presence of starch. The zone surrounding colonies indicates that the organism produced the exoenzyme amylase which hydrolyzed the starch. Gram Positive Bacilli (Spore Forming) Bacillus Clostridium - Bacterial endospores are spherical or oval intracellular objects. - All spores are heat resistant and can remain dormant and viable for very long periods. Bacillus sp. This genus contains many species ex. Bacillus anthracis, Bacillus cereus, and Bacillus subtilis. - Aerobic or facultative anaerobic and some strains are strict aerobes. - Sporulation occurs in the soil and on culture media but not in living tissue. Sporulation is often stimulated on Esculin agar media. B. anthracis - Colonies are normally large, opaque, raised, irregular, and curled. On blood agar the colonies non-hemolytic, Smooth and rough Cells gram positive spore former with square ended. - Not motile