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CARA PEMBUATAN MEDIA PADA MICROBIOLOGY Selvianti P Djarudju Specimen collection isolation 5 ³I´ s inoculation inspection identification incubation Media A microbiologist can fine tune a media for almost any purpose. General purpose media are designed to grow a board spectrum of microorganisms e.g. nutrient agar/broth, brain and heart infusion, trypticase soy agar (TSA)-casein, soy digest and (TSA)NaCl. Enriched media has complex organic substrates such as blood serum and special growth factors (vitamins, amino acids). A bacteria that requires requires complex growth factors is termed fastidious. fastidious. Enrichment media facilitates the extensive growth of particular organism. organism. Base Media Breeding base medium simple breeding that contains general substances that need by large part microorganism and put also as fundamental component to make medium breeding other. Enrichment Media The addition of blood, serum extract or tryptic soy agar will support the growth of many fastidious microorganisms. Blood agar has the addition of citrated blood to tryptic soy agar to make possible variable hemolysis which permit the differentiation of some bacteria. hemolysis: greenish brown halo around the colony ( e.g. Streptococcus gordonii or S. pneumoniae) pneumoniae) hemolysis: complete lysis of blood cells resulting in a clearing effect around the colony (e.g. Staphylococcus aureus, Streptococcus pyogenes) Non hemolytic: no change in media (Staphylococcus (Staphylococcus epidermidis, Staphylococcus saprophyticus Enriched Media Chocolate agar, a medium that gets brown from heated blood. Used for isolation of N. gonorrhoeae. Blood agar plate with bacteria from human throat. This media differentiates among different colonies by appearance 6 Selective/Differential Media A selective media contains agents that inhibit the growth of certain microorganisms and select for the growth of others. A selective media is important as a primary isolation of specific organisms. E.g mannitol salt agar (MSA) has a high NaCl concentration (7.5%) which will inhibit the growth of most microbes but will select for the growth of Staphylococcus. Staphylococcus. MacConkey agar which contains bile salts as a selective 56 Download (/download/link/cara-pembuatan-media-pada-microbiology) agent. Selective/Differential Media Bile salts is a component of feces and inhibit the growth of Gram positive bacteria and encourage the growth of Gram negative rods. rods. Other selective agents: Methylene blue and crystal All materials on our website are shared by users. If you have any questions about copyright issues, please report (/document/report/caraviolet inhibit Gram positives Selenite and brilliant green are used in media to isolate Salmonella from feces. Sodium pembuatan-media-pada-microbiology) us to resolve them. We are always happy to assist you. azide is used to isolate enterococci from water and food. The Conditions at Microbial Can Grow Optimum In A 2,072 CARA PEMBUATAN MEDIA PADA MICROBIOLOGY Medium That is: Medium must contain all easy nutrients be used by microbial. Medium must has pressure osmosis, views by selvianti-djarudju surface and pH appropriate Medium doesn't contain hindrance substance Medium must sterile General vs Selective on Apr 10, 2015 Category: Download: 0 Media Differential Media Differential media grow several types of organisms and display visible differences among Report (/document/report/caraComment: 0 DOCUMENTS organisms. Differences may show up as colony size, media colour, gas bubble formation and precipitate formation. pembuatan-media-pada-microbiology) Selective/Differential Media Mannitol salt agar is used to isolate members of the genus Staphylococcus MacConkey (/category/documents.html) agar differentiates between lactose-fermenting bacteria and (pink-red centre) and lactose-negative bacteria ( no pink 0 Share Tweet Share Share Like 0 colouration). Triple sugar iron agar (TSIA). This media contain fermentable carbohydrates Red phenol to indicate pH change Iron that indicate H2S gas production. Rxns (left to right) are: No growth Growth with no acid Acid production Comments in the bottom only Acid production in the bottom and H2S gas formation (black) Differential Media Chromagar orientation uses colour-formation to distinguish at least 7 common urinary pathogens. This allow for rapid 0 Comments Sort by Oldest identification and treatment. In this example, the bacteria were streaked as to spell their names. Differential Media Characteristic Media Characteristic media are used to test bacteria for particular activity, product or requirement. E.g. urea broth used to detect for urease. Kinds of medium is Medium of Potato Dextrosa Agar (PDA), PDB, NA, NB, LB, Add a comment... LA, TEA, AND TEB. . Pembuatan Media NA (Nutrien Agar) Untuk Bakteri Peptone««««.................................................. 5,00 g Beef Extract ..........................................................3,00 Bacteriological Agar........................................... 15,00 Final pH 6,8 ± 0,2 at 25ºC PDA (Potato Dextrose Agar) untuk Jamur Potato(solids) ...................................................... 4,00 g Dextrose............................................................. 20,00 Facebook Comments Plugin Bacteriological Agar ...........................................15,00 Final pH 5,6 ± 0,2 at 25ºC Direct Microscopic Examination Direct microscopic examination of a stained specimen is often the most rapid method for the identification of characteristics. Stains include Gram, acid fast, direct fluorescent antibody test (DFA). DFA can be used to highlight Description the presences of microorganisms in a specimen. DFA test are available for Staphylococcus aureus, Streptococcus Download Cara Pembuatan Media Pada Microbiology pyogenes, Neisseria gonorhoeae and Haemophilus influenza. Direct Examination Micrococcus luteus E. coli (white), Micrococcus luteus (yellow), Serratia marcescens (red) Serratia marcescens Direct Microscopic Examination Direct Fluorescent Antibody Test Biochemical Tests The microbe is cultured in a media with a special substrate and Transcript tested for an end product. product. Prominent biochemical tests include carbohydrate fermentation, acid or gas CARA PEMBUATAN MEDIA PADA MICROBIOLOGY Selvianti P Djarudju Specimen collection isolation 5 ³I´ s inoculation production and the hydrolysis of gelatin or starch. Many of these test in rapid miniaturized system that can detect for inspection identification incubation Media A microbiologist can fine tune a media for almost any purpose. General 23 characteristics in small cups called Rapid test. test. The info from the rapid test are input into a computer to help in purpose media are designed to grow a board spectrum of microorganisms e.g. nutrient agar/broth, brain and heart identification of the organisms. Carbohydrate Fermentation . This medium show fermentation (acid production) infusion, trypticase soy agar (TSA)-casein, soy digest and (TSA)NaCl. Enriched media has complex organic substrates production) and gas formation. formation. The small Durham tube for collecting gas bubbles. LeftLeft- right: such as blood serum and special growth factors (vitamins, amino acids). A bacteria that requires requires complex Uninoculated negative control Centre, positive for acid (yellow) and gas (open space). Growth but no gas or acid. growth factors is termed fastidious. fastidious. Enrichment media facilitates the extensive growth of particular organism. Methyl Red Test This is a qualitative test for acid production. production. The bacteria is grown in MR-VP broth. organism. Base Media Breeding base medium simple breeding that contains general substances that need by large part MRAfter addition of several drops of methyl red solution a bright red colour is positive and yellowyelloworange microorganism and put also as fundamental component to make medium breeding other. Enrichment Media The negative. Nitrate Reduction After 24-48 hrs of 24incubation, nitrate reagents are added. Left to right: Gas formation addition of blood, serum extract or tryptic soy agar will support the growth of many fastidious microorganisms. Blood (positive for nitrate reduction). positive for nitrate reduction to nitrite ( red colour). Negative control Starch Hydrolysis agar has the addition of citrated blood to tryptic soy agar to make possible variable hemolysis which permit the After incubation on starch agar, plates agar, are flooded with iodine solution. Positive test indicated by colourless differentiation of some bacteria. hemolysis: greenish brown halo around the colony ( e.g. Streptococcus gordonii or S. area around growth. Negative test indicated below. Catalase Test Place a drop of H2O2 on the culture. A positive pneumoniae) pneumoniae) hemolysis: complete lysis of blood cells resulting in a clearing effect around the colony (e.g. reaction show gas bubbles. Often used to differentiate Streptococcus from Staphylococcus. Biochemical Tests Other Staphylococcus aureus, Streptococcus pyogenes) Non hemolytic: no change in media (Staphylococcus (Staphylococcus biochemical tests of interest include: H2S production Indole test Oxidase test Oxidation fermentation Phenylalanine epidermidis, Staphylococcus saprophyticus Enriched Media Chocolate agar, a medium that gets brown from heated deaminase test Antibiotic susceptibility tests Principle, procedure, most common use. Rapid Tests Rapid test: a blood. Used for isolation of N. gonorrhoeae. Blood agar plate with bacteria from human throat. This media differentiates biochemical system for the test: identification of Enterobacteriacae and other Gram ve bacteria. It consist of plastic among different colonies by appearance 6 Selective/Differential Media A selective media contains agents that inhibit strips with 20 l of dehydrated biochemical substrates used to detect biochemical characteristics. The biochemical the growth of certain microorganisms and select for the growth of others. A selective media is important as a primary substrates are inoculated with pure cultures and suspended in physiological saline. After 5 hrs-overnight the 20 tests isolation of specific organisms. E.g mannitol salt agar (MSA) has a high NaCl concentration (7.5%) which will inhibit the are converted to hrs7-9 digital profile. Rapid Test Results OXI--0 ARA-2 + AMY-0 - 2 GLU--4 + GEL-0 VP--0 4 MEL--4 growth of most microbes but will select for the growth of Staphylococcus. Staphylococcus. MacConkey agar which + SAC-2 + RHA-1 + 7 IND--4 + TDA-0 URE-0 - 4 SOR--4 + INO-0 MAN-1 + 5 H2S--0 CIT-0 ODC-1 + 1 normal 7 digit contains bile salts as a selective agent. Selective/Differential Media Bile salts is a component of feces and inhibit the code 5 144 572 = E. coli LDC--4 + ADH-0 ONPG-1 + 5 Bacteriophage Typing Bacteriophage typing is based on growth of Gram positive bacteria and encourage the growth of Gram negative rods. rods. Other selective agents: the specificity of phage surface receptor for the cell surface receptor. Only those phages that can attach to the Methylene blue and crystal violet inhibit Gram positives Selenite and brilliant green are used in media to isolate surface receptors can cause lysis. The procedure involves: A plate is heavily inoculated so that there is no Salmonella from feces. Sodium azide is used to isolate enterococci from water and food. The Conditions at Microbial uninoculated areas. The plate is marked off in squares (15-20 mm) (15and each square inoculated with a drop of Can Grow Optimum In A Medium That is: Medium must contain all easy nutrients be used by microbial. Medium must suspension for different phages. phages. Heavily Inoculated Plate Bacteriophage Typing The plate is incubated for has pressure osmosis, surface and pH appropriate Medium doesn't contain hindrance substance Medium must sterile 24 hrs then observed for plaques. The phage type is reported as a specific genus and species followed by the types General vs Selective Media Differential Media Differential media grow several types of organisms and display visible that can infect the bacterium. E.g. 10/16/24 means that the bacteria is sensitive to phages 10, 16 and 24. Phage differences among organisms. Differences may show up as colony size, media colour, gas bubble formation and tying remain a tool for research and reference labs. labs. Unculturable Organisms Environmental researchers precipitate formation. Selective/Differential Media Mannitol salt agar is used to isolate members of the genus estimate that < 1% of microorganisms are culturable and therefore it is not possible to use phenotypic methods of Staphylococcus MacConkey agar differentiates between lactose-fermenting bacteria and (pink-red centre) and lactoseidentification. These microorganisms are called viable nonculturable (VNC). Immunological Methods The study of negative bacteria ( no pink colouration). Triple sugar iron agar (TSIA). This media contain fermentable carbohydrates antibody (Ab)- antigen (Ag) rxns in (Ab)vitro is called serology. serology. Serological rxns are the basic of Red phenol to indicate pH change Iron that indicate H2S gas production. Rxns (left to right) are: No growth Growth with immunological identification and diagnostic methods. The usefulness of serological test is dependent on its sensitivity no acid Acid production in the bottom only Acid production in the bottom and H2S gas formation (black) Differential and specificity. specificity. Sensitivity is the ability to detect minute amounts of Ab or Ag. Specificity is the ability to Media Chromagar orientation uses colour-formation to distinguish at least 7 common urinary pathogens. This allow for detect a single Ag or Ab. Ab. False Negatives/Positives High sensitivity prevents false negatives. False negatives rapid identification and treatment. In this example, the bacteria were streaked as to spell their names. Differential Media occurs when there is not reaction when the Ag or Ab is present. High specificity prevents false positives. False Characteristic Media Characteristic media are used to test bacteria for particular activity, product or requirement. E.g. positives occurs when there is cross reaction with another molecule. Precipitation Reactions Precipitation (ppt) is urea broth used to detect for urease. Kinds of medium is Medium of Potato Dextrosa Agar (PDA), PDB, NA, NB, LB, LA, the interaction of a soluble Ag with an soluble Ab to for an insoluble complex. complex. The complex formed is an TEA, AND TEB. . Pembuatan Media NA (Nutrien Agar) Untuk Bakteri Peptone««««.................................................. 5,00 aggregate of Ag and Ab. Ppt rxns occurs maximally only when the optimal proportions of Ag and Ag are present. Ppt g Beef Extract ..........................................................3,00 Bacteriological Agar........................................... 15,00 Final pH can also be done in agar referred to as immunodiffusion. immunodiffusion. Ppt test uses antibodies to detect for 6,8 ± 0,2 at 25ºC PDA (Potato Dextrose Agar) untuk Jamur Potato(solids) ...................................................... 4,00 g streptococcal group antigens. Precipitation Reactions Agglutination Reactions (Aggn) is the visible clumping of an Ag Dextrose............................................................. 20,00 Bacteriological Agar ...........................................15,00 Final pH when mixed with a specific Ab. Aggn tests are widely used because they are simple to perform, highly specific, 5,6 ± 0,2 at 25ºC Direct Microscopic Examination Direct microscopic examination of a stained specimen is often the inexpensive and rapid. Standardized tests are available for the determination of blood groups and identification of most rapid method for the identification of characteristics. Stains include Gram, acid fast, direct fluorescent antibody test pathogens and their products. products. Agglutination Agglutination Reactions Genotypic methods Genotypic (DFA). DFA can be used to highlight the presences of microorganisms in a specimen. DFA test are available for methods of microbe identification include the use of : Nucleic acid probes PCR (RT-PCR, RAPD-PCR) Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorhoeae and Haemophilus influenza. Direct Examination (RTRAPDNucleic acid sequence analysis rRNA analysis RFLP Plasmid fingerprinting. Nucleic Acid Probes A single Micrococcus luteus E. coli (white), Micrococcus luteus (yellow), Serratia marcescens (red) Serratia marcescens Direct stranded probe is added and if there is sequence complementality between the target and the probe a positive Microscopic Examination Direct Fluorescent Antibody Test Biochemical Tests The microbe is cultured in a media with hybridization signal will be detected. Hybridization is detected by a reporter molecule (radioactive, fluorescent, a special substrate and tested for an end product. product. Prominent biochemical tests include carbohydrate chemiluminescent) which is attached to the probe. Nucleic acid probes have been marketed for the identification of fermentation, acid or gas production and the hydrolysis of gelatin or starch. Many of these test in rapid miniaturized many pathogens such as N. gonorrhoeae. gonorrhoeae. Polymerase Chain Reaction (PCR) PCR is widely used system that can detect for 23 characteristics in small cups called Rapid test. test. The info from the rapid test are input for the identification of microorganisms. Sequence specific primers are used with PCR in the amplification of DNA or into a computer to help in identification of the organisms. Carbohydrate Fermentation . This medium show fermentation RNA of specific pathogens. PCR allows for the detection even if only a few cells are present and can also be used on (acid production) production) and gas formation. formation. The small Durham tube for collecting gas bubbles. LeftLeftviable nonculturables (see sensitivity table). The presence of the appropriate amplified PCR product confirms the right: Uninoculated negative control Centre, positive for acid (yellow) and gas (open space). Growth but no gas or acid. presence of the organisms. Primers are available for the identification of Niesseria gonorrhoeae, and to monitor food Methyl Red Test This is a qualitative test for acid production. production. The bacteria is grown in MR-VP broth. for the presence of Salmonella and Staphylococcus. Sensitivity of Microbe Detection Tests Methods Flow Cytometry MRAfter addition of several drops of methyl red solution a bright red colour is positive and yellowyelloworange negative. Fluorescent antibody Toxin or organism S. Typhimurium in milk Salmonella Sensitivity 103/ml in 40 min 106/ml 32 Nitrate Reduction After 24-48 hrs of 24incubation, nitrate reagents are added. Left to right: Gas formation (positive for ng/ml 1 ng/ml 1-5 cells/100 ml of H2O Latex agglutination E. coli enterotoxin ELISA PCR C. perfringens type A toxin nitrate reduction). positive for nitrate reduction to nitrite ( red colour). Negative control Starch Hydrolysis After incubation E. coli Real Time PCR and RT-PCR RT Currently many PCR tests employ real time PCR. PCR. This involves the on starch agar, plates agar, are flooded with iodine solution. Positive test indicated by colourless area around growth. use of fluorescent primers. primers. The PCR machine monitors the incorporation of the primers and display an Negative test indicated below. Catalase Test Place a drop of H2O2 on the culture. A positive reaction show gas amplification plot which can be viewed continuously thru the PCR cycle. Real time PCR yields immediate results. bubbles. Often used to differentiate Streptococcus from Staphylococcus. Biochemical Tests Other biochemical tests of results. Another application of PCR is RT-PCR (reverse trancriptase RTPCR). During RT-PCR an RNA template is interest include: H2S production Indole test Oxidase test Oxidation fermentation Phenylalanine deaminase test Antibiotic used to generate cDNA RTand from this dsDNA is generated. The enzyme used is reverse transciptase. transciptase. susceptibility tests Principle, procedure, most common use. Rapid Tests Rapid test: a biochemical system for the test: RTRT-PCR is used to detect for HIV and to monitor the progress of the disease. RTRT-PCR RAPDRAPD-PCR identification of Enterobacteriacae and other Gram ve bacteria. It consist of plastic strips with 20 l of dehydrated Random amplified polymorphic DNA PCR uses a random primer (10-mer) to generate a DNA (10profile. What are biochemical substrates used to detect biochemical characteristics. The biochemical substrates are inoculated with pure random primers? The primer anneals to several places on the DNA template and generate a DNA profile which is cultures and suspended in physiological saline. After 5 hrs-overnight the 20 tests are converted to hrs7-9 digital profile. used for microbe identification. RAPD has many advantages: Pure DNA is not needed Less labour intensive than Rapid Test Results OXI--0 ARA-2 + AMY-0 - 2 GLU--4 + GEL-0 VP--0 4 MEL--4 + SAC-2 + RHA-1 + 7 IND--4 + TDA-0 RFLP. There is not need for prior DNA sequence data. RAPD has been used to fingerprint the outbreak of Listeria URE-0 - 4 SOR--4 + INO-0 MAN-1 + 5 H2S--0 CIT-0 ODC-1 + 1 normal 7 digit code 5 144 572 = E. coli LDC--4 + ADH-0 monocytogenes from milk. PCR vs RAPDRAPD-PCR DNA sequencing The determination of a small amount of DNA ONPG-1 + 5 Bacteriophage Typing Bacteriophage typing is based on the specificity of phage surface receptor for the sequence can be used for microbial identification. The most common sequence used for microbe identification is DNA cell surface receptor. Only those phages that can attach to the surface receptors can cause lysis. The procedure sequence of the 16S rRNA gene. gene. PCR is used to amplify the 16S rRNA gene and the sequence determined. involves: A plate is heavily inoculated so that there is no uninoculated areas. The plate is marked off in squares (15-20 rRNA is a major component for ribosome and ribosome have the same function in protein synthesis in all cells. DNA mm) (15and each square inoculated with a drop of suspension for different phages. phages. Heavily Inoculated Plate Sequencing Computer analysis of 16S rRNA sequence has revealed the presence of signature sequences, Bacteriophage Typing The plate is incubated for 24 hrs then observed for plaques. The phage type is reported as a sequences, short oligonucleotides unique to certain groups of organisms and useful in their identification. rRNA specific genus and species followed by the types that can infect the bacterium. E.g. 10/16/24 means that the bacteria is sequence can be used to fine tune identity at the species level e.g differentiating between Mycobacterium and sensitive to phages 10, 16 and 24. Phage tying remain a tool for research and reference labs. labs. Unculturable Legionella. Legionella. 16s rRNA sequence can also be used to identify microorganisms from a microbial community. Organisms Environmental researchers estimate that < 1% of microorganisms are culturable and therefore it is not community. Restriction Fragment Length Polymorphism RFLP involves digestion of the genomic DNA of the possible to use phenotypic methods of identification. These microorganisms are called viable nonculturable (VNC). organism with restriction enzymes. The restricted fragments are separated by agarose gel electrophoresis. The Immunological Methods The study of antibody (Ab)- antigen (Ag) rxns in (Ab)vitro is called serology. serology. DNA fragments are transferred to a membrane and probed with probes specific for the desired organisms. A DNA Serological rxns are the basic of immunological identification and diagnostic methods. The usefulness of serological test profile emerges which can be used for microbe identification. RFLP of M. tuberculosis from 17 patients Plasmid is dependent on its sensitivity and specificity. specificity. Sensitivity is the ability to detect minute amounts of Ab or Ag. fingerprinting What is a plasmid? Plasmid fingerprinting identifies microbial species or similar strains as related Specificity is the ability to detect a single Ag or Ab. Ab. False Negatives/Positives High sensitivity prevents false strains often contain the same number of plasmids with the same molecular weight. Plasmid of many strains and negatives. False negatives occurs when there is not reaction when the Ag or Ab is present. High specificity prevents species of E. coli, Salmonella, Camylobacter and Psseudomonas has demonstrated that this methods is more false positives. False positives occurs when there is cross reaction with another molecule. Precipitation Reactions accurate than phenotypic methods such as biotyping, antibiotic resistance patterns , phage typing and serotyping. Precipitation (ppt) is the interaction of a soluble Ag with an soluble Ab to for an insoluble complex. complex. The complex
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