Microbial Taxonomy & Specimen Management PDF

MICROBIAL TAXONOMY Topic Summary ◼ Microbial classification ◼ Microbial nomenclature ◼ Microbial identification TAXONOMY ◼ An area of biological science comprising of CLASSIFICATION, NOMENCLATURE and IDENTIFICATION of organisms ◼ Provides a consistent means to organize, name and...

MICROBIAL TAXONOMY Topic Summary ◼ Microbial classification ◼ Microbial nomenclature ◼ Microbial identification TAXONOMY ◼ An area of biological science comprising of CLASSIFICATION, NOMENCLATURE and IDENTIFICATION of organisms ◼ Provides a consistent means to organize, name and identify organisms ◼ Provides a common label for every organism  1. CLASSIFICATION ◼ Organization of ◼ Taxa Designations: microorganisms that ▪ Species share similar ▪ Genus (similar species) morphologic, ▪ Family (similar genera) physiologic, and ▪ Order (similar families) genetic traits into ▪ Class (similar orders) specific groups or ▪ Division (similar classes) taxa ▪ Kingdom (similar divisions) ◼ Species ▪ Most basic taxonomic group SPECIES ▪ Collection of bacterial strains w/ common physiologic and genetic features Subspecies ◼ Subspecies – taxonomic subgroup w/in a species Biotype ◼ Biotype, serotype, genotype – groups Serotype below the subspecies level that share minor but specific characteristics Genotype Example FAMILY Micrococcaceae GENUS GENUS GENUS Micrococcus Planococcus Stomatococcus SPECIES Micrococcus luteus Example Streptococcus pyogenes Streptococcus Streptococcus agalactiae Streptococcaceae Enterococcus faecium Enterococcus Enterococcus faecalis  2. NOMENCLATURE ◼ Naming of microorganisms is based on established rules and guidelines set forth by International Code of Nomenclature of Bacteria (ICNB) or Bacteriological Code (BC) ◼ Established in 1735 by Carolus Linnaeus ◼ Binomial system ◼ Every organism is assigned a genus and species name (epithet) of Latin or Greek origin; written in italics or underlined in script ◼ Genus name – first letter is always capitalized !!!!!!!! ◼ Species name – never capitalized !!!!!!! ◼ Example: Staphylococcus aureus ◼ Informal designation – staphylococcus, streptococcus, gonococcus, etc. (not capitalized nor italicized)  Examples ◼ Staphylococcus aureus Species: Salmonella enterica ◼ Staphylococcus aureus ◼ S. aureus SUBSPECIES of Salmonella enterica 1. Salmonella enterica subspecies enterica 2. Salmonella enterica subspecies salamae 3. Salmonella enterica subspecies arizonae ◼ Escherichia coli 4. Salmonella enterica subspecies diarizonae ◼ Escherichia coli 5. Salmonella enterica subspecies houtenae ◼ E. coli 6. Salmonella enterica subspecies indica SEROTYPES of Salmonella enterica subspecies enterica 1. Salmonella enterica subspecies enterica serotype Typhi ◼ Entamoeba coli S. serotype Typhi ◼ Entamoeba coli Salmonella Typhi ◼ E. coli 2. Salmonella enterica subspecies enterica serotype Paratyphi S. serotype Paratyphi Salmonella Paratyphi 3. IDENTIFICATION ◼ Process by w/c a microorganism’s key features are delineated ◼ Identification methods ◼ Genotypic characteristics – genetic makeup ◼ DNA base composition ratio ◼ Nucleic acid base sequence base analysis ◼ Phenotypic characteristics – observable characteristics ◼ Macroscopic morphology ◼ Microscopic morphology ◼ Staining characteristics ◼ Environmental requirements ◼ Nutritional requirements ◼ Resistance profiles ◼ Antigenic properties ◼ Subcellular properties Genotypic Identification Methods Genotypic Criteria Principles Examples DNA base composition ratio 4 bases (guanine, cytosine, adenine, thymine) Example: an organism with a G/C content of 50% is related to another organism w/ the same G/C content Nucleic acid base sequence analysis Base sequence is the order of bases along a DNA or RNA strand; the extent to w/c sequences are homologous b/n 2 microorganisms can be determined by molecular methods.  Phenotypic Identification Methods Phenotypic Criteria Principles Examples Macroscopic morphology Characteristics of microbial growth pattern on culture media E.g. size, shape, texture, pigmentation of colonies Microscopic morphology Size, shape, intracellular inclusions, cellular appendages, and arrangement of cells under the microscope Staining characteristics Staining reaction/affinity to dyes as an aid to identification. E.g. Gram stain, acid fast stain Environmental requirements Ability of organism to grow at various temperatures, in the presence of oxygen and other gases, at various pH levels etc Resistance profiles Exhibition of a characteristic inherent resistance to specific antibiotics, heavy metals, or toxins by certain microorganisms Antigenic properties Profiling microorganisms by various serologic and immunologic methods Subcellular properties Establishment of molecular constituents like components of the: cell wall, cell membrane and enzymatic contents Assigned Readings: ◼ Page 6 Mahon ◼ Chapter 1 Bailey and Scott SPECIMEN MANAGEMENT Topic Summary 1. General Specimen Guidelines 2. Specimen Collection Requirements for Specific Sites 3. Specimen Transport and Processing 4. Selection and Inoculation of Primary Media 5. Incubation 6. Observation of Growth Characteristics  GENERAL SPECIMEN GUIDELINES 1. Specimen should be collected from the actual infection site. 1. Obtain cultures before the initiation of antibiotics. 1. Consider the history and physiology of the disease when considering the time of specimen collection. 1. The specimen should be in sufficient quantity. 1. Specimens must be collected with sterile collection methods. 1. Actual time between specimen collection and plating to media should be kept at a minimum. 1. A complete and accurate label is essential. SPECIMEN COLLECTION 1. Specimen should be 2. Obtain cultures collected from the before the actual infection site initiation of ▪ Contamination from adjacent tissue and antibiotics the normal flora should be avoided since ▪ Antibiotics may inhibit growth of this may overgrow the pathogen and lead pathogens, resulting to a negative to erroneous result culture, even if the patient has a bacterial infection ◼ Example: ▪ When this is not possible, it is ▪ wound infection – needle aspiration important for the physician to note ▪ Bronchitis, pneumonia – sputum any prescribed antibiotics 3. Consider the history and 4. Specimen should be in physiology of the disease sufficient quantity to when considering the time perform all procedures of specimen collection requested ▪ The causative organism may be ▪ Inadequate specimens shd be marked QNS (quantity not recovered from different sources at sufficient) and held until the different times during the course of physician determines w/c test/s illness should be performed ▪ Example: In typhoid fever, ▪ Physician should be notified if a Salmonella Typhi is present repeat collection is needed in: ▪ Blood – 1st week of illness ▪ Feces/urine – 2nd/3rd wk of illness ▪ Serum – 5th week of illness 5. Specimens must be 6. Actual time b/n specimen collected w/ sterile collection and actual plating should be kept to collection methods a minimum ▪ Contaminated containers may lead ▪ Any delay may lead to the decline of to incorrect identification the pathogen and the multiplication of normal flora ▪ if possible, cotton swabs shd not be used since organisms have a ▪ In general a 2-hour limit b/n tendency to dry out collection and processing is established for those specimens not preserved in transport medium. ▪ Swabs are NEVER APPROPRIATE for ANAEROBIC CULTURE!!! 7. Complete and accurate label ▪ Patient’s full name ▪ Hospital ID number & room number ▪ Date & time of collection ▪ Source of collection ▪ Physician’s name SPECIMEN COLLECTION REQUIREMENTS 1. Respiratory tract 2. Gastrointestinal tract 3. Urine 4. Blood 5. Cerebrospinal fluid 6. Genital tract 7. Wound cultures and abscesses Respiratory Tract Upper RT 1. Throat cultures ▪ primarily requested to diagnose group A streptococcal infection (Streptococcus pyogenes) ▪ Other suspected infections: ▪ Whooping cough – Bordetella pertussis ▪ Epiglottitis – Haemophilus influenzae ▪ Oral thrush – Candida albicans ▪ Oral gonorrhea – Neisseria gonorrhoeae ▪ Dacron/Calcium alginate-tipped swabs are suitable for collecting most URT microorganisms ▪ Most important consideration: avoid oropharyngeal flora. The posterior pharynx should be swabbed while the roof, sides of the mouth and tongue should be avoided! Respiratory Tract 2. Nasopharynx ▪ Preferred for the: ▪ dx of pertusssis ▪ dx of middle ear infections (rarely) ▪ Identification of asymptomatic carrier state of S. aureus and N. meningitidis ▪ Nasopharynx is cultured by guiding a swab through the nasal septum until the posterior pharynx is reached Respiratory Tract 2. Nasopharynx ▪ Preferred for the: ▪ dx of SARS-CoV2 ▪ Use only synthetic fiber swabs with thin plastic or wire shafts that have been designed for sampling the nasopharyngeal mucosa. ▪ Do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and may inhibit molecular tests. ▪ CDC recommends collecting only the NP specimen, although an OP specimen is an acceptable specimen type. ▪ If both NP and OP specimens are collected, combine them in a single tube to maximize test sensitivity and limit use of testing resources.  Respiratory Tract Lower RT- for the dx of bronchitis or pneumonia caused by: ▪ Mycoplasma pneumoniae, Streptococcus pneumoniae, ▪ Klebsiella pneumoniae, Serratia species, ▪ Pseudomonas aeruginosa Escherichia coli, ▪ Staphylococcus aureus, anaerobes and fungi ◼ Specimen: Sputum. It is important to obtain deep cough secretions (to avoid too much contamination from saliva). A saline gargle may enhance sputum collection. ◼ Bartlett’s Classification ▪ assessment of the quality of sputum samples; ▪ the number of PMNs and epithelial cells per LPF are enumerated; ▪ sputum scores of zero or less indicate a lack of inflammation or the presence of saliva Sputum sample Bartlett’s Classification Number of neutrophils/LPF Grade Less than 10 0 10-25 +1 Greater than 25 +2 Mucus +1 Number of epithelial cells/LPF Grade 10-25 -1 Greater than 25 -2 TOTAL SCORE Scores of zero or less indicate lack of inflammation or presence of saliva Other methods in determining the suitability of sputum sample include scanning for: 1. Squamous epithelial cells ◼ Purulent secretions contain >25 2. PMNs PMNs and 25 indicate respiratory secretions Interpretation: ◼ Few – less than 10/LPF ◼ Oral secretions are indicated by ◼ Moderate – 10-25/LPF >25 SECs/LPF and 25/lpf) neutrophils, sputum gram stain score of 0 because of 1+ (1-4/lpf) epithelial cells, abundant squamous 3+ (11-50/oif) cells and absence of Gram (+) diplococci neutrophils  Gastrointestinal Tract ◼ GIT samples are for the dx of ◼ STOOL SPECIMENS are gastroenteritis. preferred over rectal swabs. ◼ Bacterial infections of the stomach are rare due to ◼ When examining for the low pH, thus, most infections involve the ova/parasites, sample shd be intestines. analyzed ASAP! If delay is ◼ The clinical symptoms of gastroenteritis include: anticipated, sample shd be preserved nausea, vomiting and diarrhea. in polyvinyl alcohol (PVA) and buffered formalin. ◼ The nausea and vomiting usually result from the ingestion of preformed toxins instead of bacterial ◼ Direct Fecal Smear (DFS) – invasion. wbcs, parasites, yeast; high wbc count indicates bacterial infection ◼ Consider px’s history: ▪ Type and quality of ingested food/water ◼ When analysis of bacterial pathogens is ▪ Areas traveled (poor sanitation) required, an enrichment media ▪ Recent antibiotic use (selenite broth or gram negative broth) ▪ Similar problems w/ family members shd be inoculated to enhance isolation.  Rectal swab Stool specimens are preferred over rectal swabs. Urine ◼ For the dx of UTI of the upper or ◼ Specimen of choice for bacterial lower tract. culture is the clean-catch midstream urine specimen. ◼ Lower UTIs may involve the bladder (cystitis) or the urethra (urethritis). Sources ◼ Periurethral area is cleansed w/ soap, of infection: sterile water, and sterile gauze using a ▪ Normal flora coliforms front-to-back motion. ▪ Urinary catheters ▪ Contaminating bacteria from health care workers ◼ The first few mls is discarded to flush urethral bacteria. The ◼ Upper UTIs include infections of “midstream” portion is collected into a the kidney such as: sterile container ▪ pyelonephritis ▪ glomerulonephritis. ▪ pyelitis (renal pelvis and ureters) Urine ◼ A routine or randomly-voided specimen is not acceptable due to contaminants from the periurethral and vaginal areas. ◼ A catheterized specimen may be used for bacterial culture ONLY if the px cannot void. ◼ Urine is normally STERILE in the bladder area and therefore this type of urine specimen shd contain NO BACTERIA. ◼ Uncultured urine shd be refrigerated to minimize bacterial contamination. Boric acid may be added to the urine for preservation. Urine Suprapubic Urine Aspiration ◼ May be performed on infants and young children. Performed only when absolutely necessary since this is an invasive procedure. ◼ The skin is disinfected and the area is locally anesthesized. A small incision is made, and a needle is inserted into the bladder to withdraw the specimen. ◼ This procedure is reserved for pxs who cannot be catheterized when a urine sample is urgently needed. Urine ◼ A colony count is performed on all urine samples submitted for bacterial culture. ▪ greater than 1.0 x 10⁵ colonies/ml of urine indicate UTI ▪ 1.0 x 10³ to 1.0 x 10⁵ colonies/ml of urine indicate contamination or period of recovery from UTI after administration of antibiotics ◼ The most common causative agent of UTI is Escherichia coli. ◼ Staphylocccus saprophyticus is known to cause UTIs w/ colony count colony counts in a lower range of less than 1.0 x 10⁵ colonies/ml of urine ◼ Other causative agents of UTI are: ▪ Klebsiella ▪ Proteus ▪ Enterobacter ▪ Enterococcus faecalis ▪ Pseudomonas aeruginosa Escherichia coli from a urine sample  Blood ◼ 1 to 10 bacteria/ml of blood can be associated w/ bacteremia ▪ Transient bacteremia – normal flora bacteria may be introduced into the blood ▪ Intermittent bacteremia – bacteria are sporadically discharged from extravascular abscesses or infections into the blood ▪ Continuous bacteremia – constant release of bacteria into the blood from subacute bacterial endocarditis or intravascular infections ◼ The highest concentration of bacteria in the blood occurs before the fever spikes. Since this cannot be predicted, it is recommended to collect blood at 2-3 different venipuncture sites at least 1 hour apart. This serves to maximize the chances of isolating the organism. ◼ It is imperative to prepare the skin properly before phlebotomy to ensure that no skin contaminants are collected. ▪ Adults – 10 ml of blood during each phlebotomy ▪ Children - 1-5 ml of blood during each phlebotomy Blood culture tubes  Cerebrospinal Fluid ◼ Collected for the dx of meningitis ◼ CSF is collected by lumbar puncture in the 3rd or 4th lumbar vertebra by a physician. ◼ In most adults, the collection consists of 3 tubes: ▪ First 2 tubes may contain skin contaminants ▪ 3rd tube for bacterial examination Normal CSF ◼ Normal CSF is clear and colorless. Its volume, color and appearance shd be recorded upon receipt of sample. ◼ It shd be screened immediately for the presence of bacteria or fungi by methylene blue stain or Gram stain. The presence of any organism shd be reported. ◼ NO DELAY in processing of CSF since causative agent/s are usually fastidious and prone to chilling or drying (i.e. Neisseria meningitidis and Haemophilus influenzae). CSF fr a px w/ meningitis LUMBAR PUNCTURE  Genital Tract ◼ For the dx of urogenital infections: ▪ Male – urethral discharge ▪ Female – uterine cervix, urethra, cervix ◼ Causative agents for GT infections: ▪ Neisseria gonorrhoeae ▪ Herpes simplex virus type 2 ▪ Chlamydia trachomatis ▪ Trichomonas vaginalis ◼ Because of the abundance of normal flora in the female GT, use a culture medium selective for N. gonorrhoeae such as Lewis-Martin or Modified Thayer Martin (MTM). These media inhibit normal flora. ◼ N. gonorrhoeae requires chocolate agar (as culture medium) and incubation at increased CO2 concentration urethritis  Wound Cultures and Abscesses ◼ Collect specimens for wound culture using a needle and syringe to aspirate the drainage. This avoids the collection of normal flora and enhances the recovery of anaerobes which are often associated w/ deep wound infections and abscesses. ◼ Exogenous wound infections may result from: ▪ Animal and human bites ▪ Burns, ulcers ▪ Traumatic wounds (stabbing, gunshot) ◼ Endogenous wound infections – bacterial sources w/in the px ▪ Cellulitis ▪ Dental infections ▪ Septic arthritis SPECIMEN TRANSPORT AND PROCESSING SPECIMEN TRANSPORT ◼ Specimens should be transported to the laboratory w/in 2hrs of collection ◼ Specimen containers should be sealable, leak-proof, plastic bags w/ a separate section for paperwork. ◼ Bags should be marked w/ a Bio Hazard label ◼ Many organisms are susceptible to: oxygen, temperature changes, pH changes – there is a need for transport/holding media to ensure viability Holding/Transport Media ◼ maintain the viability of microorganisms in specimens w/o supporting its growth ◼ charcoal is sometimes added to absorb fatty acids (present in cotton swabs) that could kill fastidious organisms like Neisseria gonorrhoeae or Bordetella pertussis ◼ Examples: Stuart’s Medium 1. Stuart’s system- consists of swabs in a test tube w/ transport media. The buffered semisolid agar contains sodium thioglycollate that can maintain bacteria for up to 72 hours. 1. Amies Medium – modified Stuart’s medium 1. Cary-Blair medium – for the transport of stool specimens; especially recommended for the transport of enteric pathogens. Amies Medium Cary-Blair Medium Amies Transport Medium Stuart’s Medium Stuart’s Medium Anticoagulants ◼ prevent clotting like in blood, bone marrow, synovial fluid; since organisms may be bound in clots ▪ 0.025% SPS (sodium polyanethol sulfonate) – for Neisseria and some anaerobic bacteria ▪ Heparin – viral cultures; may inhibit growth of Gram positive bacteria and yeast  SPECIMEN STORAGE Storage Methods 1. Refrigeration (4C) ▪ urine, stool, ▪ viral specimens, ▪ sputa, swabs, catheters 2. Ambient Room Temperature – anaerobic bacteria 1. Body Temperature(37C) – CSF (always) 1. Freezer Temp or below -20C – serum for 1 week 1. -70C – tissues or specimens for long term storage SPECIMEN LABELING AND SPECIMEN REQUISITION SPECIMEN LABEL SPECIMEN REQUISITION ◼ Name ◼ Patient’s name ◼ Hospital number ◼ Identifying/hospital number ◼ Age/date of birth ◼ Birth date ◼ Sex ◼ Collection date and time ◼ Ordering physician ◼ Exact nature and source of specimen ◼ Diagnosis ◼ Immunization history ◼ Current antimicrobial therapy Sample Requisition Slip  Rejection of Unacceptable Specimens 1. Information on the label does not match the information on requisition slip 2. Transported at the improper temperature 3. Transported at the wrong medium 4. Quantity is insufficient 5. Leaking specimen 6. Transport time exceeded 2hrs, not preserved 7. Received in a fixative (formalin) 8. Specimen has been received for anaerobic culture from a site known to have anaerobes as part of the normal flora 9. Dried up specimen 10. Processing would produce information of questionable medical value (eg Foley catheter tip) SPECIMEN PROCESSING ◼ Gross Examination ◼ Direct Microscopic Exam ◼ Selection of Culture Media ◼ Specimen Preparation 1. Gross Examination ◼ Gross examination of specimen includes: ▪ Color ▪ Volume ▪ Appearance ◼ Areas w/ blood or mucus shd be located and sampled for culture and direct exam Bloody stool specimen ◼ Stool should be examined for evidence of Barium (chalky white color) ◼ Notations on specimen’s consistency 2. Direct Microscopic Examination ◼ Purpose: ▪ Quality of specimen can be assessed ▪ Can give an early indication on the px’s condition ▪ For comparison of what grows on culture and microscopic exam ◼ Direct examinations are usually NOT PERFORMED on: throat, nasopharyngeal, or stool specimens but are indicated from most other sources. ◼ Most common staining techniques: ▪ Gram stain ▪ Acid fast stain ▪ KOH, PAS, and calcofluor white stain (fungal exam)  3. Specimen Preparation Some specimens require initial treatment before inoculation: ◼ Homogenization – grinding of tissues ◼ Concentration – centrifugation/filtration of large volumes of sterile fluids ◼ Decontamination – i.e. sputum that may contain Legionella, Mycobacteria Swab specimens are often vortexed in 0.5 – 1ml of saline or broth for 10-20seconds to dislodge material from fibers  3. Specimen Preparation Some specimens require initial treatment before inoculation: ◼ Homogenization – grinding of tissues ◼ Concentration – centrifugation/filtration of large volumes of sterile fluids ◼ Decontamination – i.e. sputum that may contain Legionella, Mycobacteria Swab specimens are often vortexed in 0.5 – 1ml of saline or broth for 10-20seconds to dislodge material from fibers MICROBIAL CULTIVATIO N Microbial Cultivation ◼ Process of growing microorganisms in culture by taking bacteria from the infection site (in vivo) and growing them in an artificial environment in the laboratory (in vitro). ◼ The successful transition from the in vivo to in vitro environment requires that the nutritional and environmental growth requirements be met Principles of Bacterial Cultivation ◼ Microscopy provides preliminary information about bacteria involved in an infection, but bacterial growth is required for definitive cultivation and identification. ◼ Purposes of Bacterial Cultivation 1. To grow and isolate all bacteria present in a clinical specimen 2. To determine which bacteria are the causative agents and w/c are contaminants or colonizers 3. To obtain sufficient growth of clinically relevant bacteria for identification and characterization Before incubation After incubation – w/ growth Before incubation After incubation – w/ growth  Culture Media ◼ Culture medium is a mixture of the nutrients needed by microorganisms for cultivation. ◼ Most contain an energy source (carbon, nitrogen, sulfur, phosphorous, hydrogen, oxygen, and buffers). ◼ Dyes and antibiotics may also be added to Solid form either increase or decrease the viability of a particular organism or group of organisms. ◼ Culture media can be in: 1. Solid form – agar plate; red algae polysaccharide AGAROSE 2. Liquid form – broth 3. Semisolid form – w/ 0.5% or less agarose content ◼ Selection of media to inoculate is based on the organisms most likely to be involved in the disease process. Semisolid form Liquid form Why Agar? ◼ Has a unique property of melting at about 95 – 100C and solidifies at 40C ◼ Few microbes can degrade agar, so it remains solid CONSISTENCY/FORMS 1. AGAR SLANT – if the medium in the test tube is allowed to solidify in a slanted position. Eg: TSI 1. AGAR DEEP – if the medium in the test tube is allowed to solidify in an upright position. Eg. SIM 1. AGAR PLATE – if the agar is poured into a petri plate. Eg. BAP, Mac ❑ Microorganisms growing in or on such a medium form a culture. ❑A culture may be: ⮚ a PURE CULTURE if only one type of organism is present and PURE CULTURE ⮚ MIXED CULTURE if populations of different organisms are present. ◼ The culture medium prepared must be initially STERILE so that the culture will contain only the microbes added to the medium. MIXED CULTURE TERMS ◼ Inoculum – microbes introduced into a culture medium ◼ Colony – masses of microbial growth in a culture medium ◼ Agarose– a complex polysaccharide derived from Inoculation marine alga used to solidify culture media. ▪ Agar – solidified culture medium w/ agarose ▪ Agar plate – Petri dish containing agar FORMS OF CULTURE MEDIA 1. Liquid/Broth – to propagate large number of microorganisms in fermentation studies and biochemical tests Examples: ▪ Nutrient Broth (NB) ▪ Trypticase Soy Broth (TSB) ▪ Thioglycollate Broth (FT) ▪ Brain Heart Infusion (BHI) ▪ Gram Negative Broth (GN Broth) ▪ Selenite Broth ▪ Tetrathionate Broth STRICT STRICT FACULTATIVE FACULTATIVE AEROBES ANAEROBES ANAEROBES AEROBES Growth occurs Growth occurs Growth occurs Growth appears only where high only where there throughout the as “puff balls”; concentrations is no oxygen; tube; can grow e.g. Gram of oxygen have growth at the w/ or w/o positive cocci diffused into the bottom of tube oxygen medium e.g. G(-) bacilli 2. Semisolid With 0.5% or less agar content Functions: ▪ Fermentative studies ▪ Determining bacterial motility ▪ Promoting anaerobic growth Example: Rappaport – Vassiliadis (MSRV) Medium Sulfide Indole Motility (SIM) Medium  Umbrella-shaped pattern Sulfide Indole Motility (SIM) Medium produced by Listeria monocytogenes 3. Solid Media Functions: for surface growth of microorganisms; to observe colony appearance for pure culture isolations observe specific biochemical reactions Examples: ▪ Blood Agar Plate (BAP) ▪ MacConkey Agar (Mac) ▪ Eosin Methylene Blue Agar (EMB) ▪ Mannitol Salt Agar (MSA) Proteus mirabilis Chromobacterium violaceum Serratia marcescens Enterobacter sakazakii Klebsiella pneumoniae Bacillus anthracis A B C D E F  Media Classifications and Functions Type of General Isolation Non-selective Differential Selective Media Media/ Isolation Media Media Supportive Media/ Media Enrichment Media Function Support the growth of Contain a nutrient Provide distinct Allow the growth of most nonfastidious supplement colonial only one type of bacteria w/o giving required for the appearances of pathogen (normal flora advantage to any growth of particular bacteria to aid in are inhibited) group of bacteria bacteria their identification Examples: Nutrient agar Sheep blood agar MacConkey agar HE agar Trypticase soy agar (5% defibrinated (lactose fermenters SSA Nutrient broth sheep rbcs) appear pink-red, XLD while non-lactose Chocolate agar fermenters appear (heated SBA or colorless) enzyme treated to hemolyze rbcs) General Isolation/Supportive Media Nutrient Agar Enrichment Media Blood Agar Plate Hemolytic Pattern in BAP Blood Agar Plate Hemolytic Pattern in BAP Blood Agar Plate Differential Media MacConkey Agar (Selective-Differential) Lactose fermenter Non-lactose fermenter Differential Media EMB Agar (Eosin Methylene Blue)  Selective Media XLD Agar (Xylose-Lysine-Desoxycholate) TCBS Agar (thiosulfate citrate bile salts) Assigned readings: ◼ Mahon pgs 976-994 ◼ Chapter 5 Bailey and Scott ◼ Chapter 3 Delost