TYPES OF CULTURE MEDIA Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) PRINCIPLES OF CULTIVATION Grow and isolate Distinguish Obtain Grow and isolate ALL bacteria present in a clinical specimen Distinguish which isolate is PATHOLOGIC and which are contaminants Obtain sufficient amounts of bacteria to allow for identification and sensitivity Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CULTIVATION Growing microorganisms taken from in vivo infection site to an artificial in vitro environment. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) Objectives in cultivation Provide the nutritional and environmental requirements of the organisms Increase the number of organisms to make it sufficient for subsequent testing Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) TWO PHASES OF CULTURE MEDIA SOLID Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) LIQUID LIQUID MEDIA • BROTH • Nutrients are dissolved in water • Bacterial growth is indicated by TURBIDITY • 106 bacteria per mL of growth for turbidity to be seen by the naked eye. • Location of the turbidity can be presumptive of the identity of the bacteria Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) SOLID MEDIA • Nutrients dissolved in liquid with the addition of solidifying agent • AGAROSE – most common; capable of melting at high temperatures (>95) and re-solidifying only after cooling to 55 to 60C for distribution to petri dishes. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CLASSIFICATION OF SOLID MEDIA Non-Selective Selective Differential Enrichment Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) Classification of media NON-SELECTIVE – supports the growth of non-fastidious organisms IDENTIFY Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CLASSIFICATION OF MEDIA ENRICHMENT – enhance the growth of a particular bacterial pathogen from a mixture of organisms by using nutrient specificity. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CLASSIFICATION OF MEDIA SELECTIVE – contains one or more agents that are inhibitory to all organisms except those being sought. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CLASSIFICATION OF MEDIA DIFFERENTIAL – contains factors that allow colonies of one bacterial species to exhibit characteristics to distinguish them from others. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) MEDIA STERILIZATION • Timing of autoclave sterilization should start from the moment the temperature reaches 121C • Requires 15mins at 15psi • Each plate usually contains 25ml of molten agar. • Delicate media containing antibiotics and other heat-labile substances are sterilized by membrane filtration. Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) AMOUNT OF MEDIA dispensed PER PLATE/tube ❑25 ml for Small plates ❑60 ml for Big plates ❑About 5ml broth for 10ml tubes ❑About 2ml broth for 5ml tubes Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) environmental requirements: co2 and oxygen AEROBIC – grow well in room temperature FACULTATIVELY ANAEROBIC – grow in the presence or absence of oxygen MICROAEROPHILIC – survive in low levels of oxygen Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) ANAEROBIC – poor or no growth in the presence of oxygen 35-37C – optimal; most aerobic 30C – other 42C – Campylobacter jejuni 20-40C – Listeria monocytogenes; Yersinia enterocolitica environmental requirements: temperature Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) environmental requirements: ph 6.5- 7.5 (neutral) – most clinically significant bacteria; media are buffered to this range Acidophiles or acidophilic organisms are those that thrive under highly acidic conditions (usually at pH 2.0 or below) - Lactobacillus acidophilus (below pH 5.0) Alkaliphiles are a class of extremophilic microbes capable of survival in alkaline (pH roughly 8.5–11) Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) Other • CANDLE JAR – 3% CO2 • CO2 INCUBATORS – 3-5% generate CO2 at 3% humidity Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BILE ESCULIN AGAR TYPE OF MEDIA DIFFERENTIAL COMPONENT/BASE MEDIUM Nutrient agar base with ferric citrate INHIBITORS Sodium desoxycholate INDICATORS BROWN ferric citrate by group D bacteria PURPOSE Differential isolation and presumptive identification of group D Streptococci and Enterococci Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BILE ESCULIN AZIDE AGAR WITH VANCOMYCIN TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Nutrient agar base with ferric citrate INHIBITORS Azide for Gram negative bacteria, Vancomycin for resistant Gram positive bacteria INDICATORS BROWN ferric citrate by group D bacteria PURPOSE Vancomycin-resistant Enterococci Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BLOOD AGAR TYPE OF MEDIA NON-SELECTIVE COMPONENT/BASE MEDIUM Trypticase soy with 5% sheep's blood INHIBITORS - INDICATORS - PURPOSE Cultivation of Non fastidious organisms, determination of hemolysis patterns Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BORDET-GENGOU AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Potato-glycerol enriched with 15-20% defibrinated blood INHIBITORS Methicillin for contaminants INDICATORS - PURPOSE Bordetella pertussis and Bordetella parapertussis Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BRAIN HEART INFUSION TYPE OF MEDIA NON-SELECTIVE COMPONENT/BASE MEDIUM Dextrose, pork brain and heart dehydrated infusions INHIBITORS - INDICATORS - PURPOSE For non-fastidious Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) BUFFERED CHARCOAL-YEAST EXTRACT AGAR (BCYE) WITH ANTIBIOTICS TYPE OF MEDIA SELECTIVE/ ENRICHMENT COMPONENT/BASE MEDIUM Yeast extact, charcoal and salts supplemented with Lcysteine HCl, ferric pyrophosphate, ACES buffer and alpha-ketoglutarate INHIBITORS Polymyxin B for Gram-negative; Vancomycin for Gram-positive; Ansamycin for Yeast INDICATORS - PURPOSE Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH Legionella (2021) BURKHOLDERIA CEPACIA SELECTIVE MEDIUM TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone Yeast Extract INHIBITORS Bile salts, Gentamicin, Ticarcillin, Polymyxin B INDICATORS - PURPOSE Burkholderia Cepacia Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CEFSOLUDIN-IRGASAN-NOVOBIOCIN (CIN) AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone base with yeast extract, mannitol, and bile salts INHIBITORS Cefsoludin, Irgasan, Novobiocin INDICATORS Neutral Red, Crystal Violet PURPOSE Yersinia Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) CYSTINE-TELLURITE BLOOD AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Infusion agar with 5% sheep blood INHIBITORS Potassium tellurite for Gram-positive and Gram-negative INDICATORS BLACK - tellurite reduction by C.diphtheriae PURPOSE Corynebacterium diphtheriae Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) EOSIN METHYLENE BLUE (EMB) (LEVINE) TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Peptone base and lactose INHIBITORS Dyes are toxic to Gram-positive INDICATORS Eosin Y and methylene blue depending on pH (lactose fermentation) PURPOSE Differential isolation of Lactose and Non-lactose fermenting enteric bacilli Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) HEKTOEN ENTERIC AGAR TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Peptone base agar with bile salts, lactose, sucrose, salicin, and ferric ammonium citrate INHIBITORS Bile salts, dyes INDICATORS COLORLESS - Salmonella and Shigella; ORANGE/SALMON- non pathologic enteric bacilliBromthymol blue and Acid fuchsin PURPOSE Salmonella and Shigella Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) LOEFFLER'S MEDIUM TYPE OF MEDIA SELECTIVE COMPONENT/BAS E MEDIUM Animal tissue(heart), dextrose, eggs and beef serum, and sodium chloride INHIBITORS - INDICATORS - PURPOSE Corynebacterium Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) MAC CONKEY AGAR TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Peptone base with lactose INHIBITORS Crystal violet and bile salts for Gram-positive INDICATORS NEUTRAL RED INDICATOR: PINK - Lactose fermenters; COLORLESS - Non-lactose fermenters PURPOSE Differential isolation of Lactose and Non-lactose fermenting enteric bacilli Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) SORBITOL-MAC CONKEY AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone base with sorbitol INHIBITORS Crystal violet and bile salts for Grampositive INDICATORS Neutral Red indicator: PINK - Sorbitol fermenters; COLORLESS - Non-Sorbitol fermenters PURPOSE E.coli 0157:H7 Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) MANNITOL SALT AGAR TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BAS E MEDIUM Peptone base, mannitol INHIBITORS Salt concentration of 7.5% inhibits most bacteria INDICATORS Phenol Red indicator: PINK/RED PURPOSE Selective differentiation of Staphylococci Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) NEW YORK CITY (NYC) AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone agar base with cornstarch, yeast dialysate, 3% hemoglobin, horse plasma INHIBITORS Vancomycin, Colistin, Amphotericin B, Trimethoprim INDICATORS - PURPOSE Neisseria gonorrhoeae Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) REGAN LOWE TYPE OF MEDIA SELECTIVE/ ENRICHMENT COMPONENT/BASE MEDIUM Charcoal agar with horse blood INHIBITORS Cephalexin, Amphotericin B INDICATORS - PURPOSE Bordetella pertussis Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) SALMONELLA-SHIGELLA (SS) AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone base with lactose, Ferric citrate, sodium citrate INHIBITORS Brilliant green and bile salts for coliforms INDICATORS Neutral red Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) PURPOSE Salmonella and Shigella SELENITE f TYPE OF MEDIA SELECTIVE/ ENRICHMENT COMPONENT/BASE MEDIUM Peptone base broth INHIBITORS Sodium Selenite toxic for most Enterobacteriaceae INDICATORS - PURPOSE Salmonella Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) SKIRROW AGAR TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Peptone and soy protein agar with lysed horse blood INHIBITORS Vancomycin for Gram-positive; Polymyxin B and Trimethoprom for Gram-negative INDICATORS - PURPOSE Campylobacter Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) THAYER-MARTIN AGAR (modified Thayer-Martin) TYPE OF MEDIA SELECTIVE COMPONENT/BASE MEDIUM Blood agar enriched with hemoglobin and supplement B INHIBITORS Colistin, Nystatin, Vancomycin, Trimethoprim INDICATORS - PURPOSE Neisseria gonorrhoeae and Neisseria meningitidis Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) THIOGLYCOLLATE BROTH TYPE OF MEDIA NON-SELECTIVE COMPONENT/BASE Pancreatic digest of casein, soy MEDIUM broth, and glucose, reducing agents thioglycollate, cystine, sodium sulfite, semi-solid medium with low agar conc to reduce oxygen diffusion INHIBITORS - INDICATORS - PURPOSE Anaerobes, Aerobes, Microaerophillic, Fastidious Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH (2021) THIOSULFATE-CITRATE BILE SALTS-SUCROSE (TCBS) TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Peptone base with yeast extract, citrate, sucrose, ferric citrate INHIBITORS Sodium thiosulfate for Enterobacteriaceae; Bile salts for Grampositive INDICATORS Bromthymol blue: GREEN COLONIES - Vibrio spp; YELLOW COLONIES - Vibrio cholerae PURPOSE Diagnostic Bacteriology - RachelleVibrio Segismundo, RMT, MPH (2021) XYLOSE-LYSINE DESOXYCHOLATE (XLD) AGAR TYPE OF MEDIA SELECTIVE/ DIFFERENTIAL COMPONENT/BASE MEDIUM Yeast extract with lysine, xylose, lactose, sucrose, ferric ammonium citrate INHIBITORS Sodium desoxycholate for Gram-negative and Grampositive INDICATORS Phenol Red: COLORLESS - Salmonella and Shigella; RED other enterics PURPOSE Diagnostic Bacteriology - Rachelle Segismundo, RMT, MPH Salmonella and Shigella (2021)
