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7. Sample collection Universal precautions Wear gloves when examining patients or touching blood, body fluids, mucous membranes, nonintact skin and when handling items or surfaces soiled with blood or body fluids. Change gloves after contact with each patient. Wash hands immediately after gloves are removed. Wash hands and other skin surfaces immediately and thoroughly if contaminated with blood or other body fluids. Wear masks and protective eyewear or face shields during procedures that are likely to generate droplets of blood or other body fluids. Wear gowns or aprons during procedures that are likely to generate splashes of blood or other body fluids. To prevent needlestick injuries, needles should not be recapped, purposely bent or broken, or otherwise manipulated by hand. After disposable syringes and needles, scalpel blades, and other sharp items are used, place them in puncture-resistant containers for disposal. If the person collecting or handling the samples has exudative lesions or dermatitis, they should refrain from all direct contacts with patients and from handling patient-care equipment. People who are pregnant should be especially familiar with, and strictly adhere to precautions to minimize any infection risk. General concepts of specimen collection The person must have a general knowledge in pathology (seek advice prior to collecting from laboratory, samples must be appropriate for the purpose required: diagnosis, certification, surveillance, monitoring of vaccine effacacy, response to antimicrobial therapy) Samples are taken aseptically, containers used for sample collection must be sterile (if not taken aseptically the interpretation of results may be difficult because the relevant pathogen may be overgrown by contaminating bacteria) Site of the sample and expectations – sterile tissue/non-sterile tissue (determination of significance is greatly simplified if the sample is obtained from a normally sterile site, it may be unrealistic to expect meaningful answers with a sample obtained from an elementary canal unless one is looking for the presence or the absence of specific microorganisms like Salmonella, Campylobacter species) Samples are taken from the affected site(s) as early as possible following the onset of clinical signs. (imporant with enteric bacterial pathogens) Samples should be obtained from the edge of the lesions with some macroscopically normal tissue included. (microbial replication will be most active at the lesions edge) The laboratory should be informed if treatment has been started so that counteractive measures can be taken to increase the possibility of isolating bacteria. (alternative method of detection which is polymerase chain reaction will be employed) When possible, a generous amount of sample should be taken and submitted: Blocks of tissue (approximately 2 cm3) Biopsy material Several ml of pus, exudate or feces At least 5 ml of blood (for serology, so many tests can be carried out, and allows storing and testing with subsequent samples) Avoid cross-contamination between samples. (this is essential in highly sensitive amplification such as PCR needs to be used for the detection of the ethiological agent, precautions must be taken to avoid human infection if there is a zoonotic disease suspected) Clinical samples: Discharge Exudate Pus Milk Urine Feces Biopsy, etc. (live animal) Pathological samples: Organ tissue samples Aborted material (dead animal) Disease/pathological process Clinical sample Pathological sample Mastitis + + Urinary tract infection + Abscess + + Enteritis + Pneumonia + Collection of tissue samples Should be collected ASAP post mortem. (no use to submit old or partially deceased material for examination except for polymerase chain reaction) Tissues from outside the body cavities → tissues from thorax → abdominal tissues. Samples of at least 1 cm3 are collected and placed in separate sterile screw-capped jars. (water proof package) Samples in case of abortion The whole fetus and placenta should be submitted. If that is not possible – tissue samples from the fetus, a piece of affect placenta (2-3 cotyledons in case of ruminants), fetal stomach contents (in case of ruminants – abomasal), uterine discharge. (a clotted blood sample from the serological examination may give additional information) It is important to rule out zoonotic pathogens – Salmonella, Listeria, Leptospira, Brucella. Swabs and discharges Fluids > swabs (as the greater sample volume increases the likelihoood of detecting the causal organism) In large animals the short cotton swabs are generally unsatisfactory for obtaining nasopharyngeal specimens. (epithelial cells, mucous to detect respiratory diseases) Samples for agent isolation, places in sterile containers, many bacteria are suspectible to desiccation, especially when collected on a dry swab Samples should always be collectes from the site of infection Guarded swabs are necessary where misleading results could be generated due to contaminants adjacents sites. Samples from skin lesions If there are intact pustules or vesicles – disinfect the surface with 70% alcohol, allow it to dry, then aspirate the material with sterile syringe and fine needle. If the pustules or vesicles are not intact and raw surface of ulcers are seen – a swab may be taken. Biopsy of wound tissue can be taken after the superficial area has been cleaned and breathed? Skin scrapings can be done, using a blunt scalpel – the skin is scraped until blood is seen Blood samples Withdrawn into a aseptically syringe or vacutainer. Whole blood should never be frozen prior to submission to a laboratory. Blood for the isolation of bacteria should be prevented from clotting. When blood taken with syringe, care should be taken not to cause hemolysis Needle should be removed prior to expelling the sample, carefully into a sterile dry tube It is not accetable to submit blood to the laboratory in a syringe As bacteria can be intermittent it is advisable to take more than 1 sample within a 24 hour period Fecal and urine samples Feces – a sample (about the size of the end of a thumb) can be forwarded to the laboratory without transport medium. Rectal swabs often do not have enough fecal matter for agent detection, but if a swab is taken, it should be placed in a transport medium. (avoiding desiccation) The best method to collect urine is by cystocentesis, but collection by catheter or mid-stream urine is also acceptable Cystocentesis prevents the sample from getting contaminated or filled with debris from lower urogenital tract Samples from abscesses Abscesses – a pocket of pus (usually due to bacterial infection) that has developed within the tissues of the body. Several mililiters (~2) of pus should be collected, using a syringe, and/or a swab from the wall of the abscess should be taken. (not from the centre; from the edge bcs provides best bacterial results) Before sample collection, the area is shaved and disinfected with 70% ethanol or iodine solution. Anaerobic bacteria can be cultured from abcesses Samples from eyes and ears Eyes – conjunctival swab (taken gently while holding the palpebra apart), corneal scrapings (fine sterile spatule,cells should then be washed carefully into transport medium) , conjunctival biopsies. Ears – swabs are taken. Collection of mastitic milk Samples should be collected ASAP after first clinical signs are seen. Samples should be collected before treatment with intramammary or systemic antibiotics. (new sample can be taken after treatment 2-3 weeks) The udder should not be washed, unless it’s very dirty. The udder and teats must be dry before disinfection. Disinfection is done using 70% ethanol on cotton wool while paying special attention to teat sphincters. The two teats furthest from the operator are wiped first. The milk is collected from the two near teats first and then from the two far teats. The collection vial is opened immediately before the sample is taken. Do not put the cap on the floor. Teats cannot touch the vial. Remove (forestrip) three or four streams of milk from the quarter being sampled to minimize chances of sample contamination. Hold the collection vial at a 45° angle to keep debris from accidentally falling into the collection vial. Sample as rapidly as possible. The necessary amount is about 5 ml. Specimens for anaerobic culture Many anaerobes die within 20 minutes if they are exposed to oxygen. Specimens from animals that have died >4 h ago are usually unsuitable. (rapid post mortemn by the invasion of animals body from bacteria in the GI tract) Samples on ordinary swabs are usually of no value – they need anaerobic transport medium (for example, thioglycolate broth). Acceptable samples – blocks of tissue – 4 cm3; several mililiters of fluid placed in a sterile closed container. ( fill the fluid all the way up in the sample, essential for swabs) Transport of specimens The sooner the specimen is processed in the microbiology laboratory, the greater the probability of obtaining useful outcomes. Drying and exposure to a noxious atmosphere are the major factors that compromise specimens. (lead to inaccurate diagnosis, important to keep the sample moist or exclude the air) Transport media do not contain any nutrient material – microorganisms multiply poorly, but remain viable generally for at least 24-48 h. (swabs should always put in to transport media) Samples should be transported in contact with cold packs or wet ice. If transportation is delayed, most samples should be held in refrigerator temperature (+4 °C). Samples that may contain anaerobic bacteria should be delivered to the laboratory the sooner, the better, and these samples need to be inoculated into appropriate media immediately. Particularly sensitive to desiccation – Listeria, Streptococcus, Erysipelothrix, Pasteurella, Actinobacillus, Haemophilus etc. Specific requirements for Mycoplasma, Ureaplasma, Chlamydia and others. International transport of specimens International standards for the transport of infectious substances through any means of transport are based on the Recommendations of the United Nations Committee of Experts on the Transport of Dangerous Goods. Triple packaging: Outer cardboard container with required labels Leak-proof secondary container, sealable plastic bag Contains a sealable primary container with the media and absorbent material to capture any leakage within the secondary container Sample submission The labelling of samples must be cear and unambiguous. Samples must be submitted individually in separate leak-proof containers that are clearly marked, indicating the identity of the samples, animal ID and the date of collection. (container caps should be screwed together or taped to avoid leakage) The laboratory should supply sample submission forms. These forms must be completed by the veterinary practitioner. (must contain specific details of the tests required, clinical history, differential diagnosis, vaccination therapy, animal age etc) If there are some tests required that are not a part of routine investigation, the practitioner should contact the laboratory. 8. Bacteriological examination of samples General concepts Bacteriological examination of the sample involves cultivation, isolation and identification of the causative agent Only a pure culture can be successfully identified Laboratories have a general bacteriological examination plan Bacteriological examination of a sample generally takes about 3-10 days. Bacteriological examination plan includes the 5i approach: inoculation incubation isolation inspection identification 5i approach Inoculation – introducing of bacteria into an environment where they will grow and reproduce. Primary isolation done on different media using different techniques (what we did on lab work 7) Incubation – maintaining bacteria at the most favorable temperature and atmosphere for a certain amount of time. Takes 24-48h for bacteria to form colonies (that has been done to our samples as well) Isolation – separation of bacteria form a natural living mix of microorganisms; spreading them apart as far as possible to obtain separate colonies on solid media. ( we will do this in 8 lab work) Inspection – evaluating colony morphology. Observation or expected for obvious crode characteristics Identification by the use of different tests (biochemical, serological, molecular etc.) Bacterial identification and characterization Phenotypic methods: Cellular morphology – using light microscopy. Staining characteristics – Gram status, cell structures (flagella, endospores etc.) – using light microscopy. Growth characteristics – culturing requirements (oxygen, osmotic pressure, temperature etc.), colony morphology. Biochemical characteristics – oxidase, catalase, urease, nitrate and other tests. (utilising various defined media also metabolic characteristics) Give results that are a product of the expression of bacterial genes, these approaches can be succesful in bacterial identification but often require days to weeks for some pathogens Genotypic methods Commercial (complex) biochemical tests Biochemical tests are divided into traditional and modern Traditional tests usually involve preparing a medium specifically for one test, then inoculation of medium, incubation and waiting for 24h or less to see a visual result of the test In contrast to that right now there are commercially available systems that can assess metabolism of numerous substrates at a time and the results are usually compared with a database Main disadvantage of these systems is that it takes hours or days from inoculation to result interperentation, as they require metabolic activity and there is frequently phenotypic overlap among these species Phenotypic microarrays that assess metabolism of numerous substrates at a time. For identification at the species level, results are compared with the database. A few examples: API® 20E – for the identification and differentiation of bacteria belonging to the family Enterobacteriaceae. RapIDTM STR System – for the identification and differentiation of Streptococcus and related genera. VITEK® 2 – fully automated system that performs bacterial identification and antibiotic susceptibility testing. Bacterial identification and characterization Genotypic methods – genetic analysis through the use of nucleic acid probes or other molecular techniques. While phenotypic methods continue to hold place in certain settings molecular based tehniques have provided unpresidented insight into bacterial identification and typing Genotypic methods have enabled the identification of a large diversity of previously unknown taxa, the characterization of unculturable bacteria and facilitated metogenonomic studies on large and diverse bacterial communities Most molecular methods for bacterial identification is some form of DNA analysis. These methods range from relatively simple DNA amplification-based approaches (PCR, real-time PCR, random amplification of polymorphic DNA PCR) towards more complex methods based on restriction fragment analysis, targeted gene and whole-genome sequencing, and mass spectrometry. Polymerase chain reaction (PCR) PCR is an efficient and cost-effective molecular tool to copy or amplify small segments of DNA or RNA. It results in the exponential production of the specific target DNA/RNA sequences within a relatively short period. (approx 2h) This is an automated process which bypasses the need to use bacteria for amplyifying DNA, the amplified DNA segments are then separated and then analysed by gel-electrophoresis The presence of a specific fragment of a amplified DNA can be used to either identify an organism or identify a particular characteristic such as antibiotic resistance The 16S rRNA gene is often used for bacterial identification purposes because it is present in all bacteria. It has highly concerned sequences which are interperced with variable regions that are genous or species specific, bacteria can be identified by nucleotid sequence analysis of this specific gene and then they can be compared to a database with known sequences Some of the common types of PCR used in microbiology: Real-time PCR (quantitative PCR) Multiplexed PCR Reverse transcriptase PCR Matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF MS): although the technology has been used for decades to characterise complex macromolecules in chmistry labs, only recently has this technique been applied to organism identification The technique uses a smear of a colonial growth taken from solid media, which is then placed on a stainless steel target in a tin film The organism is then treated with a matrix which crystalises on the microbial film enabling a laser to distrubt microbial cells and proteins into ionazable fragments This soft ionization process occurs inside a vacuum contained in a flight tube, where the application of an electromagnetic charge allows the measurment of the time of the flight of ions MALDI TOF MS Each bacterial species generates a unique composition of ion fragments which once separated by the mass and charge in flight tube impact a detector that records the mass to charge ratio of ions generating a unique finger print that can be compared to a database of known organisms. MALDI TOF MS has become standard in many labs: Fast, reproducible, cost efficient and eliminates the need for dozens of biochemical tests to be maintained in inventory. MALDI TOF MS can be generated from a single colony with results in a few minutes that repeatable between labs allowing for database entries to be shared. Many commercial systems have thousands of database entries that include yeast and mold in addition to human and veterinary pathogens, plant pathogens and environmental bacteria Before using this method they need a pure culture Bacteriological day-by-day examination plan 1.st day Typically, on the first day the sample is inoculated on different media and direct microscopy can be done. Direct microscopy (direct smear) – can also be done by the clinician – (shape, staining characteristics) will help guide the choice of therapy before culture results are available. Staining and direct microscopy, is not always done but is very valuable because it may be the first indication that an infectious agent is present Gram stain (useful gram staining gram-bacteria can be observed and their shape, cellular contents of the sample are not readily discernible) Romanowsky-type stain (Wright’s or Giemsa) (can be used to differentiate cells in pathological specimens especially blood and bone marrow films, detecting parasites in blood, more used in cytology) Both staining methods have advantages and disadvantages Primary isolation – primary placement of clinical/pathological samples on culture media. Media plates may be streaked in any fashion as long as individual isolated colonies are produced after incubation: On blood agar On MacConkey agar – for Gram negative bacteria On CNAA – for Gram positive bacteria Other selective, differential or selective-differential media can be used. Media are incoluted with a portion of specimen, inoculation should be performed in a semi-quantitive fashion to enable a estimation of a relative abuncance of organisms in the sample, helps in interpretation of significance Colonies growing on all 4 quadrants of the petri plate indicate that there are large numbers of microorganisms in the sample, but if a sample yield in 1-2 colonies growing on the plate the significance of these colonies and the infectious aetiology of condition would be in doubt However this is pathogen dependant meaning that there are some organisms for which any growth at all is considered significant, the determination of clinical significance is aided by the cytology of a sample obtained by the infected site: for example isolation and/or demonstration numerous microorganisms from a normally sterile site without presence of inflammtory cells should be suspect. This can be explained by a contaminated collection device (contamination of inoculation loop) in the microbiology laboratory or contimated medium before inoculation 2.nd day Growth assessment: Is there any growth? On which media? Is it a pure culture? Is it a mixed culture? How many types of colonies are there? → possible pathogen is determined by specific criteria → pure culture isolation (if necessary). Usually on 2nd day there is evaluation of bacterial growth in all the different places that were used for primary isolation It is impossible to determine wheter a pure or a mixed culture will be yielded, but if there is a mixed culture a possible pathogen is determined Generally only a pure culture can be succesfully identified Assuming a pure culture has been isolated: Colony morphology evaluation by specific criteria. (on all the different media that were used for primary isolation) Microscopy – Gram staining. (gram reaction, arrangement can be seen) Rapid biochemical tests (“slide” tests) – production of catalase, oxidase, indole, coagulase etc. Inoculation of other biochemical tests; antimicrobial susceptibility testing; serological tests; bioassays & other tests (for example, molecular tests). Everything done up until this point often already gives a presumptive identifaction of bacterial genus and then to identify species some traditional or complex biochemical tests can be inoculated, plates for antimicrobial susceptibility inoculated, and other tests 3.rd day Evaluation of the different tests done on previous day (traditional biochemical tests, complex biochemical tests, antibiogram etc.). If pure culture was obtained time it takes for a bacterioogical day by day examination depends on how soon can the pure culture be obtained,and the time for examining samples for standard procedure also whether not an antibiogram or bioasace is used Usually day by day examination takes 3-10 days but in case of certain bacteria it could last even for months Interpretation of diagnostic results The quality of the diagnostic activity undertaken by the laboratory depends to a large degree on the clinical and epidemiological information supplied by the veterinary clinician. So when interpreting reports from a diagnostic microbiology lab it is important to remember that a negative A negative diagnostic report does not necessarily mean that the suspected microorganism is not the aetiological agent of the condition. There may be many reasons of the failure of laboratory to isolate and identify the pathogen: such as bacteria being overgrown by contaminats, bacteria having died on the way to the laboratory or the animal may have stopped excreting the microorganisms before the sample ws taken Detection of bacteria doesn’t always establish a diagnosis – results must be interpreted in combination with clinical signs, vaccination history, etc. Bacteria such as members of the Enterobacteriaceae are ubiquitous. Isolation of microorganisms may represent contamination of sample by feces, soil or their presence could be due to postmortemn invasion Some bacteria may be shed intermittently. A repeat sample following a negative examination report might be worthwhile. (salmonella, leptospira, myobacterium avium species or tubercolosis) Conflicting results may reflect the sensitivity of different assays. Thus a sample that is negative by isolation may be positive by PCR Teamwork aka cooperation between veterinary practice and laboratory for the benefit of the owner and pet It is the responsibility of the clinician to submit and collect appropiate samples accompanied by specific requests or adequate history It is the responsibilty of the microbiologist to interpret the results in relation to the information supplied 9. Biochemical tests General concepts Metabolism– the sum of all biochemical reactions that occur in a cell. Any substance that increases the rate of a reaction is called a catalyst. Enzymes– naturally occurring catalysts. Enzymes are proteins. Responsible for many biochemical reactions, specific to their substrate. Microorganisms carry out their pathogenicity via virulence factors, which often times are different enzymes (hyaluronidase, proteases, fibrinolysin etc.) Enzymes Can be classified based on three criteria: Biochemical class (transferases) Where they are found in the cell and their function: Endoenzymes (intracellular enzymes)– found inside the cell (e.g., transcriptase); Exoenzymes (extracellular enzymes)– secreted to the surrounding environment (e.g., collagenase releases collagen in connective tissue. Permanency/presence in the cell: Constitutive enzymes– continuously produced and are specific to a species; Adaptive enzymes– produced only when needed. For example, S. aureus produces beta lactamases when beta lactam antibiotics are used; to cleave a nutrient that is not normally used (lactase in the presence of lactose). Practical applications of bacterial enzymes In food industry– carbohydrate fermentation is used in production of dairy products, wine, beer, bread and others. In therapy– streptokinase (fibrinolysin) can be used to treat tromboembolism. In pharmacology– specific medication can be formulated that act against the bacterial enzymes. In diagnostics– to indentify bacterial species. (Each bacterial species have a spectrum of produced enzymes has proteloytic, haemotolytic, sacchorlytic and other activity, we can identify the isolated bacterial species) Bacterial enzymes are used to produce B12, amino acids such as lysein, antibiotics, organic acids and other valuable products (For example folic acid or vitamin B9 is used to snythesise proteins and nucleic acids in bacteria, to synthesise B9 bacteria use dihydrofolateredactase) Trimethopirm interfere with that enzyme, therefore the growth of bacteria are stopped The importance of biochemical tests Identification of bacteria based on structural differences is not possible. Not observable in routine microscopy The shape, size and arrangement of bacteria only help in the process of identification. Many species of bacteria that have similar size, shape and arrangement Each bacterial species has a distinct enzymatic profile, therefore identification can be made based on the differences in their biochemical activities. Biochemical tests could be divided the genotypic and traditional biochemical tests (we talk about traditional) Once a pure culture isolation is obtained the results from a few comparatitevly simple tests, such as as catalase or oxidase tests can often identify a bacterium to a certain level Possible identification scenes for gram positive cocci Haemolytic activity The ability for bacteria to hemolyze blood (partially or completely) indicates pathogenicity. Haemolysis patterns can help differentiate and identify certain bacterial pathogens Haemolysis is seen under and/or around the bacterial colonies (only on blood agar!): Alpha haemolysis– caused by H2O2, hemoglobin is oxidized to methemoglobin; Beta haemolysis– caused by exotoxin (enzyme) – streptolysin Look at lecture 4! Helps with diagnosing pathogenic staphylococci, streptococci, pathogenic E. coli strains and other haemolytic bacteria. CAMP test Christie–Atkins–Munch–Peterson test The CAMP test is used for the presumptive identification of Group B beta-hemolytic streptococci (Streptococcus agalactiae). The basis of the CAMP test is the enhanced haemolytic activity between beta haemolysis producing strains Staphylococcus aureus and the extracellular protein (CAMP factor) produced by S. agalactiae. Positive result produced also with: Listeria monocytogenes and Staphylococcus aureus Listeria ivanovii and Rhodococcus equi Corynebacterium renale and S. aureus. Important that these streaks do not cross in agar surface, some space is left between them Catalase test Detects the enzyme catalase that converts H2O2 (hydrogen peroxide) to water and gaseous (molecular) oxygen. 3% H2O2 is needed. Catalase positive organisms rapidly produce bubbles when exposed to a solution containing hydrogen peroxide Key in differentiating many gram positive organisms for example stapphylococci catalase positive, streptococci and enterococci catalase negative End product of normal carbohydrate metabolism and is toxic to bacteria and the production of catalase is considered a virulence factor There are two methods: A drop of H2O2 is placed on a slide and a loopful of bacterial colony is taken from a medium (other than blood agar) and placed in the drop. (Bcs RBC also contain catalase so a false positive test result may occur) Hydrogen peroxide is found aerobic and anaerobic facultative bacteria not in ANAEROBES The H2O2 is added to a colony on a plate. Blood agar should be avoided, because presence of red blood cells lead to a false positive. Coagulase test Differentiates the species of the genus Staphylococcus into two groups– coagulase positive staphylococci and coagulase negative staphylococci. Coagulase causes a clot form when bacteria are incubated with plasma Coagulase enzyme interacts with the fibrinogen converting it to fibrin. Makes a coating around bacteria which helps them escape phagocytosis Two types of coagulase– free coagulase and bound coagulase: The bound coagulase is called the clumping factor and is detected rapidly by a slide test. The free coagulase is detected in the test tube as a result of the formation of a clot. Positive Staphyloccus aureus, pseudointermedius, Negative staphyloccus epidermidis Bound is found in bacterial cell wall it can be detected with a slide method Free coagulase is secreted outside the cell wall which can be seen when the bacteria are cultivated in a tube containing plasma, a clot is formed after few hours It it important to mention that slide is used more rarely than tube method, if slide negative a tube test is incoluated anyway Oxidase test Cytochrome c oxidase is detected. Is a part of electron transport and nitrite metabolism in some bacteria, enzyme can accept electrons from artifical substrates, in this case reagent that is used in the test. Producing a dark oxidised product differentaiting between groups of gram negative bacteria Pseudomonas aeruginosa oxidase positive E.coli oxidase negative All aerobic (facultative anaerobic) bacteria use the electron transport chain (ETC), but their ETCs are not all identical. Some bacteria have cytochrome c oxidase – the last enzyme, which transfers electrons to oxygen, in electron transport chain. Anaerobes are oxidase negative. Oxidase test is a rapid test performed on a filter paper. A reagent (usually 1% tetramethyl-p-phenylene-diamine solution) is used. Carbohydrate fermentation Most organisms obtain their energy through bioxidation of a substrate carbohydrate metabolism. Organisms use carbohydrate differently depending on their enzymes, some use fermentation. Sugar fermentation can be anaerobic (glucose) or aerobic process. Facultative anaerobes are enzymatically competent to use both aerobic or anaerobic pathways. Glucose is degraded to pyruvic acid. Pyruvate metabolism results in a variety of end products (for example, lactic, formic, acetic acids, hydrogen or carbon dioxide production etc.) Carbohydrates and alcohols undergo anaerobic dissimilation and produce an organic acid such anorganic acid that may be accompanied by gaseous hydrogen or carbon dioxide A typical carbohydrate fermentation medium contains: Nutrient broth ingredients (or the support for the growth of all organisms) A specific (and just one) carbohydrate that serves as as a substrate for fermentating capabilities(glucose, sucrose, lactose, maltose, mannitol etc.) pH indicator (for example, phenol red red at neutral ph changes colour to yellow at acidic ph) Carbohydrate fermentation that ends in acid production will cause phenol red to turn yellow (positive reaction). Acid production can be accompanied by gas formation– Are visible as bubbles to detect this Durham tubes (inverted small tubes) are used. No reaction is seen if the culture is not able to ferment the carbohydrate. (Tubes red no gas production Sometimes the culture uses peptones for energy source, producing alkaline end products. Phenol red turns to deep red color. Kligler’s Iron agar and Tripple sugar iron agar tests Both of these agars are used to differentiate among the groups or genera of Enterobacteriaceae. (Gram negative bacilli capable of fermenting glucose and production of acids)Differentiation is made through differences in carbohydrate fermentation patterns and hydrogen sulfide production. Kligler’s iron agar contains two sugars– lactose and glucose, but Tripple sugar iron agar contains lactose, glucose and sucrose. Both agars contain phenol red and sodium thiosulfate (substrate to H2S). Kligler’s Iron agar and Tripple sugar iron agar tests No need for test Glucose oxidation/fermentation (O/F) test Glucose oxidation/fermentation test is used to determine how the bacterium metabolizes a carbohydrate such as glucose. Oxidative bacteria form acids from metabolised carbohydrates, produce carbohydrates only under aerobic bacteria Fermentative bacteria produce acid under aerobic and anerobic conditions Anerobic conditions are ensured with mineral oil layer over the medium in one of the tubes Medium contains also ph indicator bromathymol blue, which is neutral in green turns yellow in acidic If the bacterium is nonsaccharolytic and uses pepton as the source of energy alkaline end products are produced and it turns blue Utilization of amino acids Some bacteria are able to deaminate and decarboxylate amino acids by the use of enzymes deaminases and decarboxylases are vital in utilization of amino acids and metabolism of nitrogen compounds Decarboxylases are adaptive enzymes and are produced in the presence of specific amino acid substrates (lysine, ornithine, arginine). Three decarboxylase enzymes that are used differntiate members eneterobacteriaceae, the decarboxylase enzyme activity is determined by cultivating nutrient mediums The used nutrient medium contains glucose, the specific amino acid substrate and the pH indicator (bromothymol blue). If the bacterium ferments glucose, (decarboxylation occurs ph if the medium becomes alkaline despite the fermentation of glucose) it creates acidic environment, in which the decarboxylase (if produced) activates. End products of decarboxylation are alkaline Urease test Urease produced by some organisms Especially helpful in the identification of Proteus spp. Although other organisms may produce urease their action on the substrate urea tends to be slow, slower than seen in proteus species Therefore this serves to distinguish genus lactase fermentatating nonlactating enteric microorganisms Urease is an hydrolytic enzyme that (that cleaves ammic compound such as urea) acts on urea and forms the alkaline end product ammonia. Urease is detected in Christensen’s agar, which contains urea and pH indicator (phenol red). Presence of ammonia creates alkaline environment and that causes phenol red to turn to a deep pink color. IMViC IMViC are a group of tests that are useful in distinguishing members of the family Enterobacteriaceae: Indole test Methyl-Red (MR) test Vogues-Proskauer (VP) test Citrate test Indole test Tryptophan is an essential amino acid(which can be metabolised) and the enzyme tryptophanase into indole and pyrovic acid, ammonia mediates the conversion of tryptophan into indole. (Not charatcteristic in all microorganisms can be used in identifaction of and can be seen with kovac reagent produces a cherry red colour in case of positive result Indole can be dected in two ways: Tube test – a tube containing tryptose water is inoculated with the culture and after inoculation Kovac’s reagent is added. (Broth culture and colour chage is observed at meniscus) Spot test using filter paper, test culture and Kovac’s reagent. (Two reagents used benzaldehyde gives red colour when result is positive and the cinnamalaldehyde which gives a blue or green positive, pink for negative) Methyl red test Used to determine if the organism is able to produce stable acid end products from glucose fermentation. In the first 24h glucose is converted to pyruvic acid by all members of the Enterobacteriaceae → positive MR reaction. During prolonged incubation (2-5 days) MR positive organisms metabolize pyruvic acid to lactic, acetic and formic acids by mixed acid pathway → acidic pH is maintained. In case of a positive result, MR-VP broth becomes red after methyl red (pH incicator) is added. Vogues Proskauer test Used to determine if the organism produces a neutral end product of glucose metabolism – acetylmethylcarbinol If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, 40% KOH (strong alkali) and atmospheric oxygen. In case positive result broth becomes pinkis4h -ish-ish - ish red 6 drops of alpha napthol and 2 drop koh MR-VP test Most Enterobacteriaceae demonstrate one or the other metabolic pathway but rarely both. But onlyt one of these tests will be positive Citrate utilization test Citrate utilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source. (Can be used for distinguish coloforms such as klebsielle aerogenesn which are citrate positive and e.coli which are negative) For this test Simmon’s citrate agar is used and it contains bromothymol blue as the pH indicator. Citrate metabolism results in alkaline end products, therefore in case of a positive result, the medium color changes from green to blue.

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