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MICR_111 MR. NATHANIEL VERON CASAMINA, MSMT, RMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON #4: CULTURE ME...

MICR_111 MR. NATHANIEL VERON CASAMINA, MSMT, RMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON #4: CULTURE MEDIA AND INOCULATION OUTLINE CULTURE MEDIA ACCORDING TO CONSISTENCY Agar will melt if the temperature reaches 80 to 90° C and I. Definition of Terms solidify at 40 to 50° C. A. Types of Culture Based on 55 to 60° C = cooling temperature for distribution of agar Components into plates. B. Culture media according to Molten agar in the amount of 20ml to 25ml should be Consistency transferred to sterile plates. C. Culture media according to Agar is a sulfated polymer made up of D-galactose, Composition 3,6-anhydro-L-galactose, and D-glucoronic acid. D. Culture media according to how the medium is Dispensed/Distributed. Table No. 1 Culture media according to Consistency E. Culture media according to Use LIQUID MEDIUM SEMI-SOLID SOLID MEDIUM F. Inhibitory substances MEDIUM G. Ways to facilitate anaerobic cultivation It contains 0% agar. It contains 0.5 to It contains 2-3%. II. Inoculation techniques 1% agar. A. Streaking It allows the growth Agar is usually Examples: Triple B. Placement of fluid specimens or of aerobes, derived from red sugar iron (TSI) agar, swabs into culture broth anaerobes, and algae. MacConkey agar C. Types of inoculation facultative (MAC), Blood agar D. Measuring bacterial growth anaerobes. plate (BAP), and E. Growth on agar plate Chocolate agar plate F. Growth on agar slants (CAP). G. Growth on broth and gelatin stabs Examples: TSB, Examples: Sulfide H. Growth BHI, and indole motility Thioglycollate. (SIM) medium. DEFINITION OF TERMS Cultures - are growth of microorganism on a culture CULTURE MEDIA ACCORDING TO COMPOSITION medium. Culture medium - is a liquid, semi-solid or solid medium Table No. 2 Culture media according to Composition utilized to observe growth patterns of microorganism as SYNTHETIC OR NON-SYNTHETIC TISSUE CULTURE well as for transport and storage. DEFINED MEDIUM OR COMPLEX MEDIUM o Consistency of the culture medium is defined MEDIUM by the concentration of the agar placed on It is a medium where It is composed of It is used for obligate the culture media. all the components some unknown intracellular bacteria o Used to cultivate microorganisms and are known to the substance (Rickettsia and observe their growth patterns. user. (peptones, meat Chlamydia) o Appearance of colonies,phenotypic and yeast extracts. characteristics, color of the colonies, and some of them can be used to differentiate It is designed for It is very useful for Examples: W138 bacteria according their lactose fermentation research purposes isolation of cells, HeLa 229 cells, adn hemolytic pattern. as either a liquid or bacteria. and McCoy cells. solid medium. TYPES OF CULTURES BASED ON COMPONENTS It is preferred for the Examples: Nutrient HeLa 229 cells are 1. Pure Culture - It is composed of only one species. isolation of broth (NB) human cervical tissue 2. Mixed Culture cyanobacteria and medium, TSB, and cells while McCoy ○ It is composed of more than one species. chemoorganotrophs. MAC agar. cells and W138 cells ▪ Growth is known as mixed colonies. are fibroblasts. All of 3. Stock Culture these are used for the ○ It is composed of several species contained isolation of in a separated culture medium (one Chlamydia. species/culture medium). An example is the Embryonated eggs ○ It is used for academic and industrial BG-11 medium. are utilized for the purposes. propagation of ○ Usually termed as preserved. Rikketsia. ▪ For example we have a bacteria Staphylococcus aureus and if you want to preserved it then you should use stock culture. 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #4: CULTURE MEDIA AND INOCULATION CULTURE MEDIA ACCORDING TO HOW THE MEDIUM IS DISPENSED/DISTRIBUTED. 1. Plate Medium are distributed into the dish or plate. ○ Examples: MacConkey Agar, Blood agar plate, Chocolate agar plate, 2. Tube Medium are prepared as either liquid, slant, butt, and slant with butt. ○ Example: triple sugar iron. ○ Slant agar - Simmons Citrate Agar (SCA) ○ Deep/Butt - Sulfide indole motility agar (SIM) 4. Differential media ○ Slant with Butt - Triple sugar iron (TSI) agar ○ These media allow the visualization of and lysine ron agar (LIA). metabolic differences between groups of Examples: SIM, Triple sugar iron (TSI) agar, Simmons’ bacteria.s citrate agar (SCA), and lysine ron agar (LIA) ○ Examples: McConkey, BAP, EMB, and HEA ○ MacConkey - differentiates lactose fermenters (pink colonies) from non-lactose fermenters (colorless colonies) ○ BAP - differentiates hemolytic pattern of Streptococci ○ EMB - differentiates (E. Coli) greenish metallic sheen colonies from other lactose fermenting CULTURE MEDIA ACCORDING TO USE bacteria (dark purple colonies) 1. Simple Media/ General purpose media/ Supportive ○ HEA - identifies the Salmonella and media Shigella (bluish green colonies with ○ Are routinely used in the laboratory and black centers) from Shigella (green without added supplement. colonies) while the other coliforms ○ Are media that support the growth of most appear orange or salmon pink non-fastidious bacteria. colonies. ○ These are made up of meat and soybean ○ CLED agar also characterizes extracts. those bacteria that utilize lactose ○ Examples: Nutrient agar (NA) and broth, (both yellow colonies and medium), Trypticase Soy Broth (TSB) and it is recommended for urine 2. Enrichment media (Liquid-type media) culture. ○ It is used to propagate the growth of certain groups of organisms. MAC CONKEY AS DIFFERENTIAL MEDIUM ○ They contain specific nutrients; liquid-type media. Neutral Red (pH indicator) provides differntial test. ○ It can also be used as supplement to agar Bacterial fermentation of lactose results in acid production plates to detect aerobes, anaerobes, and which decreases pH and causes the neutral red indicator microaerophiles. to give bacterial colonies a pink to red color. ○ These media are incubated for a certain period and then subcultured to isolate the BAP - HEMOLYTIC PATTERNS desired microorganisms. Alpha/Partial/Incomplete Hemolysis ○ Examples: Alkaline peptone water, Selenite ○ “Greenish zone of hemolysis F., Thioglycollate, Tetrathionate, Beta/ Complete Hemolysis Gram-negative (GN) broth, Lim broth (group ○ “Clear” zone of hemolysis B streptococci) Gamma/ No Hemolysis 3. Enriched media and Non-selective media ○ These are media with added supplements 5. Selective media such as blood vitamins and yeast extract, ○ These are media incorporated with necessary for the growth of fastidious antibiotics, dyes, or chemicals to inhibit the organisms. growth of other organisms while ○ These are solid-type media promoting the growth of the desired ○ Examples: BAP and CAP organism. ▪ BAP is the standard nonselective ○ Examples: MacConkey agar, Hektoen culture medium. enteric agar (HEA) (Selective and Differential medium), Xylose Lysine Deoxycholate agar ▪ CAP with horse blood is used for the (XLD), BSA and Mannitol salt agar (MSA) isolation of Haemophilus. (7.5% salt - Can be toxic to some organisms, but also has halophilic organisms, ex. Staphylococcus) ○ Has inhibitory substances that prevents the growth of other organisms and promote the growth of target organisms. 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03  LESSON #4: CULTURE MEDIA AND INOCULATION NOTES TO REMEMBER Anaerobic condition - 0% O 5-10% CO. 5-10% H. + HE agar contains bile salt and dyes (acid fuchsin) for 80-90% N. (anaeröbe jars, bags or chambers) non-enteric pathogens; it inhibits the normal flora of the lower GIT. INOCULATION TECHNIQUES XLD agar contains 0.25% sodium desoxycholate salt and sodium thiosulfate to differentiate Salmonella (red with STREAKING black centers) from Shigella (red or clear colonies), Most commonly performed inoculation technique General-purpose isolation streak - yield a INHIBITORY SUBSTANCES semiquantitative estimate of growth (4 quadrants) 1. Inhibitory to Gram-positive bacteria: crystal/gentian 4th quadrant yield and where isolated colony is present, violet (present MacConkey agar), basic fuchsin, and isolate the pure colonies in the culture media bile salt 2. Inhibitory to Gram-negative bacteria: potassium tellurite and sodium azide 3. Inhibitory to swarming bacteria (proteus species): alcohol and chloral hydrate 6. Special Media o Is used to isolate bacteria with specific growth requirements. o Examples: Löwenstein–Jensen medium (has egg and malachite green that facilitates the growth of MTB, the colony has a cauliflower appearance) and Thiosulfate-citrate-bile salts-sucrose agar (special media use for isolation of vibrio cholerae, fermentation and produce yellow color colony) STREAKING FOR ISOLATION Perform aseptic technique each and after quadrant NOTES TO REMEMBER (sterilize loop and culture media) The selection of media to inoculate is based on the type of No need to sterilize when done in Biosafety cabinet specimen submitted for culture and the organism likely to Pure isolated colonies can be seen in the 4th quadrant, it be involved in the infectious process. is use to perform bacterial identification for biochemical If several plates will be inoculated for a given specimen, testing and antimicrobial susceptibility testing for the media should be arranged beginning with the antibiotics susceptible or resistant bacteria most enriched medium and progressing to the most selective - inhibitory substances added to selective media will not be carried over from one agar to another. Primary plated culture media - the standard in hospital First they will plate it in a Blood agar plate using the 4 quadrant streaking, get colony and transfer to chocolate agar plate and after streaking, get colony and then transfer to Macconkey agar plate. Table No. 3 pH Indicators Phenol Bromth Neutral red Bromcr Red ymol esol Blue Purple PLACEMENT OF FLUID SPECIMENS OR SWABS INTO Acidic Yellow Yellow Yellow Yellow CULTURE BROTH Environment Place a few drops of the liquid specimen into the tube Alkaline Red Blue Red Purple/ Place the swab into the broth. Environment Violet o Example: Trypticase soy broth - Placing the Found in: TSI, HEA, MacConkey LIA swab directly to the broth to transfer the XLD, SCA, , bacteria in, get the loop and can then be use MSA, TCBS Salmonella to start plating in the culture media. Christen Shigella o The broth is an example of media that is an sen’s Agar (SSA) enriched broth media use to facilitate the Urea growth of the organism Agar TYPES OF INOCULATION WAYS TO FACILITATE ANAEROBIC CULTIVATION 1. Stabbing Use of special culture medium - thioglycollate ○ This technique is applied in Sulfide, Indole, Boiling of culture medium Motility Agar (SIM) Use of anaerobic chamber system ▪ SIM - Inoculating needle is used Use of Gaspak jar or Kendall jar. instead of using inoculating loop Plastic bag or pouch containing calcium carbonate and (stab directly in the SIM medium) catalyst ○ It is also used to inoculate streptococci into BAP. 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #4: CULTURE MEDIA AND INOCULATION ▪ Stab in BAP to observe the GROWTH ON AGAR PLATE hemolytic pattern of the streptococcus spp. ○ Only used when determining the hemolytic pattern of strep. or the sulfide production of Enterobacteriaceae spp. 2. Stab and Streak (Slant Media) ○ It is used to inoculate organisms into and onto triple sugar iron (TSI) agar and lysine iron agar (LIA). ○ 2 preparation: Butt portion of TSI and Slant portion of TSI ○ Get the colony and directly stab it to the butt portion of TSI, move it upwards and streak. 3. Pour Plate Technique ○ Is a technique where the organism is added GROWTH ON AGAR SLANTS to a molten agar, mixed and then allows the medium to solidify. ○ You cannot isolate colonies perfectly in pour plate technique GROWTH IN NUTRIENT BROTH AND GELATIN STABS Growth in nutrient broth REMEMBER o Formation of pellicle, turbidity, and sediment is an Inoculating Loop- Sterilized and cooled thoroughly example of bacterial growth Specimens on swab - roll and streak Growth in gelatin stabs Bedside or direct inoculation of specimen to culture o Filamentous or swirling/straight line pattern of media is optimal for isolation of the pathogen bacteria Urine specimens (quantitative isolation) - a calibrated loop is used (0.01mL or 0.001 mL) MEASURING BACTERIAL GROWTH Colony Count (Colony Forming Unit/mL) Number of colonies x Dilution Factor = CFU/mL 1 ul = factor 1.000 ( 0.001 mL) 10 uL = factor 100 (0.01 mL) Example: o Number of colonies = 170 colonies o Dilution factor = 1,000 o 170 colonies x 1,000 = 170,000 CF/mL or 1.70 x 105 CFU/mL 4 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #4: CULTURE MEDIA AND INOCULATION GROWTH Table No. 4 Culture media and their purposes 1. Cell Count - Microscopy, Electronic Particle Counter, Culture Medium Purpose Colony Count Alkaline peptone broth Vibrio ○ Neubauer counting chamber - can also be Anaerobic blood agar (CDC) Obligate anaerobic bacteria used for counting RBCs and WBCs aside Bacteriodes bile esculin agar Gram-negative anaerobes from bacteria Bile esculin agar Enterococci and group D Microscopic Count Plate Count streptococci Bismuth sulfite agar Selective media for Salmonella (stool) Blood agar Primary plated medium; Differentiation of hemolytic patterns Bordet-Gengou agar Bordetella pertussis and Bordetella parapertussis Brilliant green agar Selective medium for Salmonella Blood cystine dextrose agar Francisella tularensis Brain heart infusion agar Fastidious organisms and broth ○ Cell counter - For easy visualization of Buffered charcoal yeast Legionella spp. and counting bacterial volume extract (BCYE) Nocardia spp. Campy-blood agar Campylobacter SPp Campy-thioglycollate broth Selective holding medium for Campylobacter, used for cold enrichment technique (4°C) CDC anaerobe 5% sheep Obligate, slow-growing 2. Cell Mass - Weighing, Measurement of Nitrogen blood agar anaerobe content, Turbidimetry Cefoperazone vancomycin Campylobacter Spp. ○ Membrane/Molecular Filters amphotericin (CVA) agar Cefsulodin-irgasan-novobioc Yersinia enterocolitica and in (CIN) agar Aeromonas Cetrimide agar Pseudomonas aeruginosa Chick embryo Chlamydia and Rickettsia Chocolate agar Fastidious bacteria such as Haemophilus and pathogenic Neisseria Chocolate agar with horse Haemophilus Spp. blood Chromogenic media Methicillin-resistant Staphylococcus aureus (MRSA) Columbia colistin-nalidixic Gram-positive cocci acid agar (Columbia Note: Anaerobic CNA for CNA) anaerobes Cystine tellurite blood agar Corynebacterium (CTBA) diphtheriae Cycloserine cefoxitin Clostridium difficile fructose agar (CCFA) Czapek agar Nocardia asteroides ○ Turbidimetric Method (Cell Mass) - Without Dorset egg medium Mycobacterium tuberculosis turbidity all light will be transmitted and Nocardia asteroides Deoxycholate citrate agar Salmonella and Shigella (stool specimen) Dieudonne's medium Vibrio cholerae Dubos oleic agar Mycobacterium tuberculosis Edward-Hayflick agar Mycoplasma Eosin methylene blue or Differential medium for Levine's medium lactose fermenters (LFs) and non-lactose fermenters (NLFs) Ellinghausen, McCullough, Leptospira interrogans Johnson, and Harris 3. Cell Activity - Biochemical Activity (EMJH) medium 5 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #4: CULTURE MEDIA AND INOCULATION Fletcher semi-solid medium Leptospira Thayer-Martin agar Neisseria gonorrhoeae and Fetal bovine serum agar Haemophilus ducreyi Neisseria meningitidis with vancomycin Thioglycollate broth Aerobes, anaerobes, Footpads of mice (living Mycobacterium leprae microaerophiles, and tissue) aerotolerant anaerobes Gram-negative broth Selective and enrichment Thiosulfate-citrate-bile Vibrio medium for enteric salts-sucrose (TCBS) agar pathogens like Salmonella Tinsdale agar Corynebacterium and Shigella diphtheriae Human blood Tween bilayer Gardnerella vaginalis Todd-Hewitt broth with Enrichment and selective medium nalidixic acid and medium for Streptococcus Hektoen enteric (HE) agar Differentiation of Salmonella gentamicin or colistin agalactine and Shigella Tryticase soy broth (TSB) Blood culture bottle base Kelly medium Borrelia burgdorferi and enrichment broth Laked Bacteroides and Prevotella Tyrosine agar Aerobic actinomycetes Kanamycin-Vancomycin with Xylose lysine deoxycholate Salmonella and Shigella Blood (LKVB) (XLD) agar agar Leffler blood serum medium Corynebacterium diphtheriae Löwenstein-Jensen (LJ) Mycobacterium REFERENCES agar Lysine iron agar Enterobacteriaceae 1. Notes from the discussion by SIR NATHANIEL (deamination or VERON M. CASAMINA, MSMT, RMT, ASCP decarboxylation) Litmus milk media Clostridium spp. MacConkey (MAC) agar Differential medium for LF and NLF; selective for Gram-negative bacteria MacConkey (MAC) agar with E. coli 0157:H7 in fecal sorbitol specimen Mannitol salt agar Staphylococcus aureus Martin-Lewis agar Neisseria gonorrhoeae McBride medium Listeria monocytogenes Methylene blue milk Enterococcus Middlebrook 7H10 agar Mycobacteria Modified Lombard-Dowell Aerobes and anaerobes medium Modified Thayer-Martin Agar Neisseria gonorrhea and Neisseria meningitidis Mueller-Hinton agar Susceptibility test (antimicrobial testing) New York City (NYC) agar Neisseria gonorrhoeae, Ureaplasma urealyticum and Mycoplasma spp. Oxidative-fermentative (OF) Differential media for medium non-fermentative bacilli Petragnani medium Mycobacteria Phenylethyl alcohol (PEA) Selective isolation of aerobic medium and anaerobic Gram- positive bacteria Rabbit blood agar with yeast Haemophilus influenzae extract Regan-Lowe agar Bordetella pertussis Salmonella-Shigella agar Salmonella and Shigella (SSA) Schaedler agar Non-selective medium for aerobes and anaerobes Seller agar Pseudomonas aeruginosa Selenite broth (enrichment Salmonella Spp. medium) Skirrow agar Campylobacter spp. Tetrathionate broth Salmonella and Shigella spp. 6 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03