Bacterial Cultivation PDF - Medical Technology

lOMoARcPSD|12161157 Bacterial Cultivation Medical Technology (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Clandesti...

lOMoARcPSD|12161157 Bacterial Cultivation Medical Technology (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 BACTERIAL CULTIVATION - And if bacteria are getting it from organic compound, most of the common organic compound that we use in micro bio- laboratory are Carbohydrates like glucose, lactose and Included in this topic are: sucrose. - If they’re capable silang kuhanin ng carbon from this organic  Nutritional requirements compound, we call them heterotrophs  Environmental requirements  Heterotrophs are known as consumers because  Bacterial growth phase they consume producers or other consumers. Dogs,  Different culture media birds, fish, and humans are all examples of heterotrophs. Bacterial growth - Btw, most of the normal flora are called heterotroph. - Refers to the increase in the number of bacteria rather than in  Nitrogen size. - Basically bacteria in number and do not grow size. - This growth is affected by various factors such as optimum Nitrogen- needed for synthesis of proteins growth requirements, dynamics of growth, including the use of a medium that can be artificially prepared in the laboratory.  Free nitrogen from the air  Nitrogenous compounds in the culture media (e.g. In bacterial growth, it refers to the growth of number, dumadami sila. peptone, yeast, beef extract) Not about the size. - is for making proteins and nucleic acid. Nitrogen makes a 40% of the dry weight bacteria. - Nitrogen ay makikita sa atmosphere, from the air, but if let’s say nutrition for example mga anaerobic, anaerobic, hindi pwede icontact from the air because of the oxygen. So kailangan natin maglagay or magdagdag ng mga Nitrogenous Bacterial compounds like peptone, beef extract and yeast. growth environment  Energy When you grow or cultivate bacteria, there are several nutritional Energy source and environmental factor that they need for them to successfully multiple, increase in number  Light- phototroph  Chemical energy- chemotroph Each bacteria have different sets of nutritional and environmental factor requirements. - Energy is in the form of ATP, Adenosine 5′-triphosphate. Para makapagperform ng metabolic functions, ng cellular functions. Nutritional Requirements - So now let’s talk about Phototroph. So some of bacteria are There are 3 major nutritional needs for bacterial growth. CNE: capable of getting energy from light. We call them Carbon, Nitrogen and Energy phototroph. - For chemical energy, let’s say for example some are getting it from organic compounds and capable of converting it into  Carbon energy, we call them chemotroph. - Now some bacteria require smaller amount of molecules such Carbon- Needed for the synthesis of cellular components as phosphate, phosphate is for nucleic acid. Phospholipids also of the cell membrane. And then  Carbon dioxide from the air- autotroph sulphur, for protein synthesis. The 3 smaller amount of  Organic compounds in the culture media (glucose)- molecules is additional 4% of dry weight bacteria. heterotroph - is needed to make cellular constituents or cellular components;  Electron sources it represents almost 50% of the dry weight of a bacterium. This is one of the major requirement. Basically eto yung pinaka Electron source kailangan niya out of all the requirements. - So what could be the sources of carbon? Carbon could be from  Inorganic molecule (Fe2+) – lithotroph the carbon dioxide.  Organic molecule- organotroph - So when bacteria are getting its carbon from carbon dioxide  NADH reduced from NAD that that group of bacteria, we call them autotrophs.  FADH2 reduced from FAD  Autotrophs are known as producers because they - , so if the bacteria are capable of getting that electron are able to make their own food from raw materials from inorganic molecules we call them lithotrophs. and energy. Examples include plants, algae, and some types of bacteria. - If they get the electron from organic molecule, we call them organotroph. Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 - We also have Nicotinamide Adenine Dinucleotide - Where can we found the hemoglobin? This are found in the (NADH) and Flavin Adenine Dinucleotide (FAD). So RBC, this is a protein component of red blood cells. when electron are needed, na - rereduce sila. There are 2 most common type of culture media that has x and v factor. So now let’s talk about CAP (chocolate agar plate)  Water/moisture/humidity - Lysed RBC (heat)this characteristic can lead to the availability Humidity- we have to remember that bacterial cell is 70% water, or non-availability of the x and v factor meaning it’s like a sealed plate of moisture. Therefore humidity level This heat will inactivate the NADase and therefore pag should be maintained at 70%. nainaactivate, magkakaron ng NAD, at pag may NAD tayo, magkakaron ng V factor. Pano yung namemaintain sa laboratory? So you have to put a - Ang mangyayari sa CAP dahil nagapply tayo ng heat, container of water inside the incubator, we have to monitor it malalysed si RBC. At dahil don sa inapply nating heat, regularly para hindi magdry out. And also need icheck daily kasi according to chemistry, mamamatay ang mga enzyme because possibility is pwede magkamold or fungi na pwede maggrow doon sa of the heat. Because of that, the NAD itself will not be dis- incubator which could be contaminants of out cultures. May mga activated also or hydrolysed kaya magiging present si V factor. incubators naman na equipped na with humidity regulator. - It has both X and V factor Both of them contains blood  mineral elements BAP ( Blood agar plate) needed as co-factors in various metabolic process of the bacteria (e.g. Ca2+, Mg2+, Fe2+, Sulfates, Phosphates) - Intact RBC - No v factor, only x factor is available - Other than the ones I’ve mention earlier, some bacteria are - NADase available kaya si NAD mahahydrolysed, si V factor, capable of needing a cofactors. Usually yung mga nagiging mawawala. cofactors na yon, mineral elements. For example, - For example there is a red cell, so intact ang RBC natin. So first calcium, magnesium, sulphate, phosphates. Not all, some need meron tayong enzyme na tinatawag na NADase. NAD is is mineral elements. hydrolysing the NAD. NAD is V factor. So therefore, ang nangyayari, nawawalan tayo ng V factor, because it is hydrolysed or degraded by this enzume, kaya ang natitira lang  Salt ditto ay si X factor. bacteria can tolerate salt concentration below 6%, howeverthere are What about if kailangan natin ng.. uhm kunwari wala tayong BAP. certain bacteria that can survive high salt concentration environment For example, haemophilus it requires both x and v, what to we do. hence they’re called as Halophilic bacteria or Halophiles (salt- Gumagamit tayo ng BAP pero need natin both, so meron tayong loving) tinatawag na satelitism. For salt, hindi lahat kaya magtolerate ng salt, however may mga bacteria na nagsusurive at high salt concentration called HALOPHILIC bacteria or HALOPHILES (salt-loving) Examples of bacteria who loves salt???? Hahahaha For example this is BAP. So ang nangyayari ditto, kapag macuculture tayo ng suspected haemophilus spp, surrounding them  Staphylococcus spp- usually grown in MSA, MSA has are another culture of organisms. Ang usually na ginagamit dito is 7% sodium chloride. staphylococcus aureus- meron tong tinatawag na  Enterococcus spp. betahaemolithic factor or B- hemolysin. And this could be  Vibrio spp. except Vibrio cholerae & Vibiro mimicu- up a source of the V factor. This can liberate the V factor. Pwedeng to 10% salt concentration gamitin si BAP as long as may pagkukunan tayo ng V factor.  Bacillus spp. San nanggagaling yung RBC? Maglalagay talaga tayo dapat ng Additional requirement, there are bacteria which are very difficult to blood. Usually yung mga sources ng blood natin is equally 5% grow, we call them fastidious organisms. defibrinated sheep blood. We also get it from horse blood, from rabbit blood and even human blood. But for human blood, it should Example the x factor and the v factor. be TYPE O. type o has non- specific inhibitor. - X factor, also known as hemin or hematin - V factor is v for vitamins, technically (Nicotinamide Adenine Dinucleotide or NAD) - The haemophilus spp requires this. Haemophilus are blood loving organism, some of them requires both, some of them requires only x or v factor. Lagi dapat may X or V or both factors. Environmental Requirements - Kadalasan kapag nagcuculture ng haemophilus, nilalagyan ng blood yung culture medium kasi X factor or thee hemin is from - Gas hemoglobin. This is a degradation product of hemoglobin. - Temperature Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 - pH- optimal pH for bacteria or bacterial growth is neutral of  Aggregatibacter spp., slight alkaline environment.  Cardiobacterium spp.,  Eikenella spp. Gaseous requirement  Kingella spp. - Environmental requirements, we get this from the atmosphere.  Streptococcus pneumonia Like oxygen, carbon dioxide. Groups of organism Temperature requirement Aerobe - most pathogenic bacteria would grow at temperature between 35-370C, hence incubator in the laboratory is usually set and - bacteria that grow, live, and survive in the presence of oxygen maintained within this temperature range for routine isolation of pathogens. Strict/ obligate aerobe  Mesophilic- 20-40 degree Celsius  Psychrophilic/Cryophilic- bacteria growing optically 0-20 - absolutely requires oxygen to grow, live, and survive. degree Celsius - Micrococcus spp., Mycobacterium spp., Pseudomonas spp.,  Thermophilic- 50-60 degree Celsius Neisseria spp., Brucella spp., Francisella spp., Bordetella spp.,  Hyperthermophilic/Extremely Thermophilic- 80-110 degree Leptospira spp. Celsius (spore forming bacteria)  Eurithermophilic- growing at a wide range of temperature Facultative anaerobe  Sternothermophilic- usually growing at a narrow range of temperature. - bacteria that have the ability to grow, live and survive in small concentration of oxygen environment pH requirement - pwedeng wala, pwedeng meron. They may survive with or even a small amount of oxygen. - most pathogenic bacteria can grow in a neutral or slightly - Staphylococcus spp., Streptococcus spp., Family alkaline environment (pH 7.0 – 7.5), hence most culture media Entrobacteriaceae used in routine isolation of pathogens is adjusted to this pH range. Microaerophilic  ACIDOPHILIC – acid loving bacteria (e.g. Lactobacillus acidophilus) - bacteria that prefer small concentration of oxygen environment  ALKALIPHILIC – alkali loving bacteria (e.g. Gardnerella approximately 2%- 10% vaginalis) - Usual oxygen available sa environment is 18% of oxygen.  HIGH OSMOTIC PRESSURE – OSMOPHILIC Bacteria - Campylobacter spp., Helicobacter spp., Arcobater spp., and (Archaebacteria spp.) some streptococcus spp. - Etong mga example na to is kailangan ng mas mababa. BACTERIAL GROWTH PHASE Anaerobe Refers to the stages of bacterial growth - Bacteria that grow, live, and survive in the absence of oxygen. Strict/ obligate anaerobe - Absolutely do not require oxygen to grow, live and survive. - Strict means wala, 0% talaga, dapat walang oxygen - Many Clostridum spp. such as Clostridium noyvi, most Bacteroides spp.,Fusobacterium spp., Peptostreptococcus spp., and Porphyromonas spp. Aerotolerant anaerobe - Bacteria that do not require oxygen but may tolerate or withstand limited exposure to oxygen - Some Clostridium spp. such as Clostridium perfringens, Bacteroides fragilis, moststrains of Proprionibacterium and Lactobacillus Capnophilic (capnophiles) 1. Lag phase - Bacteria that requires 5-10% of carbon dioxide for growth. 2. Logarithmic growth phase - Usual amount of carbon dioxide sa environment ay 1-3% lang 3. Stationary phase pero meron iba na nagrerequire ng mas mataas na amount ng 4. Death phase carbon dioxide and this are the following:  Sa x-axis is yung duration, yung time.  (NHACEK Group)  Sa y-axis yung logarithm is yung dami ng viable cells  Neisseria spp., (buhay na cells)  Haemophilus spp.,  Ang bacteria, they are replicating via binary fission. Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4  Binary fission, is an example of a sexual reproduction. Bacteria are still adjusting to Meaning to say, yung isang cell nadivide into two cells the new environment hence there is no cell division that  Meron tayong tinatawag na generation time/ Lag phase occurs in this phase but None None doubling time, This is the time required for one cell to they are activity divide into two cell. The generation time of a bacterium in synthesizing DNA and proteins that are necessary a culture media can be from 20 minutes. Example ng mga for cell division mabibilis ay e-coli. Phase where there is a  up to 24 hours, ito naman yung mga mababagal. Slow sudden increase of bacteria because of rapid generation growing bacteria like micro bacterium tuberculosis Logi/logarithmi or doubling time. The c number of generation per /exponential hour is called growth Yes Yes Lag phase phase rate constant. This is also the phase where bacteria are most - nagaadjust palang sa environment yung mga bacteria. Like us metabolically active hence students, this is our first time na magtake ng professional most susceptible to the action of antimicrobial course so nagaadujust tayo. Ganon din si bacteria, pag nilagay agents. mo sya sa culture medium, sa isang environment, magaadujust After essential nutrients are muna yun to the new environment. Maximum depleted, toxic products - So may nangyayari na bang cell division at this time? Wala pa. stationary accumulate, or oxygen /plateau phase becomes limiting, the rate of Yes No pero meron nang cellular activity. Nagsisynthesize na sila ng cell division equates rate of mga protein, ng mga DNA. cell death - Etong mga sinysynthesize nila na mga cellular components, it Due to unfavourable Decline/death environment for growth, is needed for the cell division. phase bacterial cell division Yes No - I repeat, walang cell division na nangyayari, wala ring decreases which cell death nangyayaring increase in number becomes more accelerated. Logarithm phase Growth rate - This curve or in this table makikita natin na from the lag phase, biglang umaangat ang logarithm natin as it continues to grow. Growth rate natin sa lag phase, ang pagpipilian natin is 0, +, -. - Dito na dumadami yung bacteria, rapid generation, rapid doubling time.  Lag phase- 0 growth rate - Usually yung number of generation, number of generation per  Logarithmic- + growth rate hour we call it growth rate constant.  Maximum stationary- 0 growth rate - So this is also the phase where most of the bacteria ay  Decline phase- (-) growth rate metabolically active. - Eto yung pinaka susceptible sila sa antimicrobial agent. CULTURE MEDIA Maximum stationary/plateau phase - an artificial preparation in the laboratory which contains basic foundation of nutrients and a solidifying agent (if needed) to - This is where nutrients are starting to deplete. Nauubos na support the growth of microorganisms. ang nutrients. - Additional substances may be added to enrich the media for - They are starting to produce toxins, and the toxins itself are growth of microorganisms that are very difficult to grow very harmful for microorganisms. That is why ang rate ng cell (Fastidious) division natin dito, it weights to the rate of cell death. - Artificial culture medium na merong nutritional at environmental - Meron paring nangyayreng cell division pero hindi na factors which contains basic foundation of nutrients and dumadami yung number. naglalagay tayo ng solidifying agents. Pero hindi lahat ng bacteria nilalagyan ng solidifying agents. Para msupport yung Decline/death phase growth ng microorganisms. - Nagdadagdag din tayo minsan ng additional substances kung - So dahil sa unfavourable environment, hindi na nagkakaron ng kailangan lalong lalo na if yung organisms is fastidious bacterial growth. - Culture media is mimicking (kinokopya) yung environment at nutrition na originally kung natatandaan meron tayong mga sources ng bacteria, meron tayong reservoir, origin. halimbawa nakikita sa environment, water, soil, air. Meron ding nangaggaling from na human body and even from the animals. - Kung ano man yung nutrition at environment na nakikita ntin or nirerequire nila pag tumutubo sila and those invornments, kailangan natin imimick yon artificially sa laboratory - Kaya naman iba iba ang culture medium natin, iba iba yung culture media natin dipende kung sino ang patutubuin natin. Bacterial growth phase Growth phase keynotes Cell Increase divisio in n number Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 cooling starting 42 degree Celsius and magmemelt ulit upon boiling. - SEMI-SOLID – a culture medium that contains 0.5%-1% agar.  Basically ito ay liquid na nilagyan ng.5-1% agar.  Commonly ang guston natin malaman is yung motility.  Other than motility, we can also observe the hydrogen sulphide dust production  Kadalasan nakikita sa tube TERMINOLOGIES:  Sulfide Indole Motility (SIM) Medium – used for observation of hydrogen sulfide gas production, indole production,and motility. Growth of microorganisms noun  Usually ginagamit entero bacterium show 1. CULTURE verb To grow/cultivate - SOLID – a culture medium which contains 1.5-3% (2-3%) agar. microorganism  Makikita ang solid in plated medium and even in tube. 2. INOCULATE/PLANT/CULTIVATE - Introducing the microorganism to the culture media 3. TRANSPLANT/SUBCULTURE CLASSIFICATION OF CULTURE MEDIA ACCORDING TO MANNER - Transfer of microorganisms from one CM to another. OF DISPENSING/FORMATION - Halimbawa, from nutrient agar to macConkey agar. - PLATED – usually contained in a container that can be made CLASSIFICATION OF CULTURE MEDIA of glass (pyrex) or disposable plastic.  Sa plate, it can be made or glass or sa disposable plastic petri CLASSIFICATION OF CULTURE MEDIA ACCORDING TO dishes COMPOSITION:  Ang common na ginagamit na petri dish is 100mm (standard) yung nasa laboratory. Approximately 100mm in terms of - Synthetic/Chemically Defined – composed of known diameter and exact amounts of pure chemical substances  150mm is used for anti-susceptibility testing (AST)  For bacteria. Autotrophic microorganism - TUBED – usually container in glass tubes such as Wassermann tubes with different volume capacity (3mL, - Non-synthetic/Non-Chemically Defined/COMPLEX 5mL, 10mL) or in a tube with a flat bottom and a screw cap – composed of complex materials that are rich in vitamins and  May iba’t ibang volume capacity for tube, so you have to nutrients that are not usually represented by a chemical formula consider first kung ano yung capacity ng tube mob ago ka such as peptones, beef or yeast extract, plant extracts, etc maglagay ng culture medium at bago iorient.  Combinations of chemicals and vitamins  May iba’t ibang orientation pag nilalagay sa tube, when we  For bacteria say orientation, merong slant, slant/deep or butt slant, the butt only (deep), broth (liquid) - TISSUE CULTURE MEDIA - live cells harvested from organs of humans and animals that supports the growth of  Slant, butt slant, butt is solid obligate intracellular organisms that cannot grow in artificially prepared culture media.  Kadalasan na nilalagay dito is mga viruses. Table 7-4 tissue culture media used in microbiology laboratory Tissue culture Source media Vero Cell Line - BOTTLED – culture media contained in a glass bottle that is Kidney cells of an African green monkey Mc Coy Cell Line Mouse cell line usually used for blood culture Chicken Embryo Fertilized chicken egg  This is according to brand, meron tayong BACTE ALERT A549 Cells Human lung carcinoma (pinaka common bottled) may kasama ng sps, an anti- HELLS Cell Line Human cervical carcinoma Hep-2 Cell Line Human epithelial cells of larynx carcinoma coagulant for blood. CLASSIFICATION OF CULTURE MEDIA ACCORDING TO CLASSIFICATION OF CULTURE MEDIA ACCORDING TO FUNCTION/USE PHYSICAL STATE/CONSISTENCY - LIQUID – a culture medium that doesn’t contain a solidifying GENERALPURPOSE/PRIMARY/BASIC/BASAL/SUPPORTIVE/GE NERAL ISOLATION CULTURE MEDIA agent - contains basic nutritional requirements to support the growth of  The nutrients are dissolve in water, meaning walang non-fastidious microorganisms. solidifying agent, walang nagpapatigas.  Basic nutrients ang laman para masuportahan ang non-  Laging sa tube nakikita fastidious and the reason we call is basal kasi it is a base  When we say solidifying agent, most common solidifying medium in preparation of other cultute media. Halimbawa agent na ginagamit natin is agar/agarose. yung enriched, general purpose siya na dinagdagan ng  Why agar? The reason is it is not easily degraded by bacteria additives, additives kadalasan mga special requirements ng and second is that it can be melted and can resolidify upon bacteriqa. Ex. Nagdagdag ng blood sa hemophilus kailangan Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 ng RBC. Basically BAP and CAP ay example of enriched culture  Halimbawa mcconkey kapag lactose fermenter (LF) kulay meida niyan pink. kapag Non-lactose fermenter (NLF) colorless. So  Ano po example ng general culture media? Ao yung ginamit we can use differential medium for that para maaidentify natin at first sa activity natin yung yellowish lang is the natin yung isang bacteria, or group of bacteria nutrient agar (NA) so meron din nutrient ne broth (TSB, TSA)  Selective differential, maaring ang isang culture mediuam at  This is also used a base medium in the preparation of pwedeng tawagin na both selective and differential. other culture media  Important, not all selective culture media are differential but - ENRICHED CULTURE MEDIA – contains the basic all differential are selective nutritional requirements to support the growth of non- fastidious microorganisms with additives, enriching substances, or supplements to support the growth of fastidious microorganisms. It is important to remember and understand that NOT ALL selective  general purpose siya na dinagdagan ng additives, additives culture media are differential but ALL differential culture media are kadalasan mga special requirements ng bacteriqa. Ex. selective. Nagdagdag ng blood sa hemophilus kailangan ng RBC. Basically BAP and CAP ay example of enriched culture meida INHIBITORS in CULTURE MEDIA - ENRICHMENT BROTH – a primary media used to support or favour the selective growth of pathogens in a specimen, Kung gusto mo patubuin ang gram negative then you have to add such as stool or sputum, where the number of normal flora inhibitors for gram positive bacterias outnumber the pathogens.  Kadalasan sa specimen ng mga normal flora, example si stool, INHIBITORS for GRAM POSITIVE BACTERIA - DYES – crystal violet, eosin, methylene blue, brilliant green, ilalagay kay APW, ano yung hinahanap natin na organism? If the etc. specimen is watery then we will APW, kasi watery stool is a - CHEMICALS – bismuth sulfite, bile salts (sodium characteristic stool of vibrio. desoxycholate), thiosulfate, citrate, etc.  APW is mataas an g salt concentration at mataas rin ang pH - ANTIBIOTICS – Vancomycin (inhibitor for gram positive) alkaline po and this is concentrated by vibrio spp.  So kung meron mang ibang organism, syempre may mga normal Selective from gram (-) flora sa stool, for example si e-coli, , si enterococcus, so hindi po niya msususportahan yung growth netong mga organisms ni APW INHIBITORS for GRAM NEGATIVE BACTERIA kasi nga si APW mataas ang salt concentration, mataas din ang - DYES – basic fuchsin and thionine for Brucella abortus pH. - CHEMICALS – potassium tellurite, sodium azide, phenylethy  So hindi inhibitor ang salt and pH, kumbaga mas favourable lang alcohol siya para sa growth ng gusto mong pathogen. Kasi kung inhibitor - ANTIBIOTICS – Colistin, Nalidixic Acid, Trimethoprim na yung idinagdag mo, ang tawag na don selective. (Proteus) - TRANSPORT CULTURE MEDIA – a primary isolation Selective for gram (+) culture media which maintains the viability of bacteria without INHIBITORS for FUNGI allowing rapid multiplication if there is an anticipated delay in bringing the specimen collected bedside or remotely to the - ANTIBIOTICS – Nystatin, Anisomycin, Ampothericin B. laboratory. Nung pinasa yung mga normal flora, pinafamiliarize yung mga  Minemaintain natin yung viability ng bacteria hindi normal flora per part of the body. Therefore merong mga normal flora pinagmumultiply halimbawa sa mouth meron tayong candida diyan so kung yaw  Ginagamit pag may inaanticipate na delay sa transporation or natin sumama yung candida, maari tayong maglagay ng antibiotics. delay sa collection. Halimbawa sa ibang lugar ginawa, malayo sa laboratory. INDICATORS in CULTURE MEDIA  Example is amie’s (w/ charcoal) and stuart’s transport medium- - Dyes and Chemical Substances such as pH indicators Neisseria spp  We can also and indicators, indicator cannot be inhibitor.  - transgrow and JEMBEK ( a brand) jembek means John E. Martin Indicator can be an indicator for an specific reaction. Biological, Environmental Chamber. Halimbawa ang pinakacommon na indicator na ginagamit is  Cary-blair- vibrio spp. pH indicator so we can detect either acid, alkaline products. - SELECTIVE and DIFFERENTIAL CULTURE MEDIA Kasi we have to remember ang mga bacteria  Selective culture media- favors the growth of the nagmemetabolize sila ng mga substances halimbawa organism of interest using inhibitors added in the culture kailangan nila ng mga carbon, kung naaalala niyo ang mga media heterotrophs, are normal flora in our body.  Differential culture media- contains indicators  Ano yung common source ng carbon nila? Organic which changes in color as a result of a product produced compounds such as carbohydrates. So kapag be a chemical reaction in the components of the media namemetabolize nila, nafeferment, btw sa metabolism dalawa such as glucose. yan, for bacterial metabolism they could ferment or oxidize  May dalawang characteristics na makikita, halimbawa compound. Pag sinabing ferment, without oxygen. Pag oxidize merong mixed na organism. Sa isang specimen, iba iba yung with oxygen. bacteria. Ang hinahanap natin is a lactose fermenter. Ang  Kadalasan sa mga organic compound, ang product as acid or gagawin natin gagamit tayo ng culture media na kayang alcohol. Ngayon kung ang nilagay natin is carbohydrates, halimbawa si organism kaya niya magferment ng lactose magdifferentiate yung mga kayang magferment ng lactose at (hindi lahat ng bacteria) so ang hinahanp natin is yung kayang mga hindi kayang magferment ng lactose. Kadalsan kulay ang magferment ng lactose. So pano natin malalaman if tinitingnan dito. Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 nagferment ng lactose? Kailangan makadetect tayo ng acid, kaya need ng pH indicator.  Halimbawa naman may mga organism na kaya magproduce ng hydrogen sulfide gas, ito ay colorless gas. So pano malalaman na may naproduce na hydrogen sulfide gas, so may indicator din tayo, the H2S indicator. That is to indicate if Differentiation of Family Enterobacteriaceae based on Lactose may hydrogen sulphate gas production. Fermentation  Hindi ito inhibitor, dinadagdag natin to sa kahit anong culture media, whether its selective, differential. Specially nilalagay ito Rapid RLF – Escherichia (nasa picture, ang tawag diyan ay sa differential, ang indicators. greenish metallic sheen, they produce medium size, dark violet colonies with the characteristics of greenish metallic sheen) EXAMPLES OF ENRICHMENT BROTH: Enterobacter (usually medium size, dark violet with or without dark center) , Klebsiella (large colony, mucoid dark violet with or - Alkaline Peptone Water (APW) - used to selectively without dark center) favor the growth of Vibrio while inhibiting all other normal intestinal flora due its high pH. - Meron mga rapid lactose fermenter na characteristic colony,  For vibrio, because of the high pH, high salt concentration upon growing in ENP. Malalaman na Escherichia kapag may which is unfavourable for the normal flora of the intestine, so it may be favourable of vibrio greenish metallic sheen) - Selenite F Broth - used to selectively favor the growth of - Usually within 18-24 hours, magpoproduce sila ng dark violet Salmonella while inhibiting all other normal intestinal flora.  For salmonella Late LLF - Hafnia, Serratia, Citrobacter - Thioglycolate Broth (THIO) – an enrichment broth for anaerobic bacteria but should be used solely in the isolation of - 36-72 hours pero parehas sila nagpoproduce ng dark violet anaerobic bacteriasince it can also grow aerobes and - Salmonella arizonae facultative anaerobes. - Shigella sonnei  Enrichment na ginagamit sa unaerobic bacteria na kadalasan - Yersinia enterocolitica tumutubo sa bottom ng thioglycolate broth - GN Broth (Gram Negative Broth) – used to selectively None NLF - ALL Salmonella except S. arizonae favor the growth of Salmonella and Shigella while inhibiting all other normal intestinal flora. - ALL Shigella except S. sonnei - Todd-Hewitt Broth – a liquid enrichment recommended for - ALL Yersinia except enterocolitica the production of Streptococcal haemolysin ( a virulence - Proteus factor of streptococcus )and the cultivation ofstreptococci - Providencia prior to serological grouping. Merong test para igroup yung mga - MorganellaEdwardsiella ibat ibang klase ng streptococci. MOST COMMONLY AND ROUTINELY USED SELECTIVE & DIFFERENTIAL CULTURE MEDIA IN THE LABORATORY 1. EOSIN METHYLENE BLUE (EMB) AGAR  Original Color – dark violet  Selective For: gram negative enteric bacilli  Kadalasan enterobacteria ceae  Inhibitors: dyes gram (+) eosin and methylene blue  Differential INDICATORS: eosin and methylene blue 2. MC CONKEY (MAC) AGAR - May carbohydrate ito, para maging differential siya, kailangan  Original Color – light pink natin ng lactose  Selective - Si lactose ay ginagamit na carbon source and differential For: Gram negative enteric bacilli medium or differential component kasi hindi lahat ng bacteria Inhibitors: crystal violet, bile salts, citrate ay capable magferment ng lactose that is why ang Inhibitors para sa mga gram positive enterobacteria ceaeay pwedeng icategorize as lactose  Differential: lactose fermenter (LF), late lactose fermenter (LLF) and non-lactose INDICATORS: neutral red usually acid: red/dark pink. if fermenter (NLF) no acid, it will become colourless, kapag alkaline, ang - Kapag gumagamit ng organic compound, may mga bacteria na kulay niya yellow. kaya ang fermentation, may mga bacteria na kaya ang Kapag ang carbohydrate, naferment ng bacteria ang oxidation. Most of the enterobacteria ceae, they are used in fermentation meaning even without the use of oxygen, they can product ay acid, so bababa ang pH. Kaya kailangan still use or utilized organic compounds such as lactose. natin ng pH indicator. In this case, neutral red. - Anong product? Acid. Anong nangyari sa acid? bababa Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 kleibsiella. So san papatak si salmonella and shigella? Dito sila ngayon sa colorless na colony. - If there’s H2S, it’s salmonella. If without, it’s shigella. RFK: EKE, without H2S blackening/ walang H2S production Escherichia coli and enterobacter aerogenes are EKE of RLF. 4. HEKTOEN ENTERIC (HEA) AGAR - Kapag may acid, syempre mga lactose fermenter yon, ang kulay ay dark pink.  Original Color – dark green - Kung Rapid, 18-24hrs. Kung Late, 36-72hrs  Selective: - Kung non-lactose fermenter, it will become colorless. kasi For: Gram negative enteric bacilli Inhibitors: bile salts, citrate walang acid na naform  Differential: merong lactose. - Remember ang neutral red, usually acid: red/dark pink. if no INDICATORS: pH indicator Bromthymol Blue (BTB); in acid, it will become colourless, kapag alkaline, ang kulay niya other references, bromothymol blue yellow. H2S Indicator: ferric ammonium citrate Sulfur Source: sodium thiosulfate 3. SALMONELLA-SHIGELLA (SSA) AGAR - Original color is dark green, para sa mga enteric bacilli pa rin to, meaning to say yung mga enterobacteriaceae.  Original Color – light orange - Kaya magdetect ng H2S gas production. So blackening parin, kasi  Selective: pareho ng ginamit na components. ferric ammonium citrate and For: Salmonella (H2S gas producers) meaning sodium thiosulfate. nagpoproduce ng hydrogen sulfide gas. and Shigella spp. - Ang color ng acid kapag bromthymol blue is orange. Kapag walang Inhibitors: Brilliant Green, Bile salts, Citrate acid, usually green. Kapag alkaline, ito yung nagiging blue.  Differential: INDICATORS: pH indicator: neutral red H2S Indicator: ferric ammonium citrate Sulfur Source: sodium thiosulfate - Iniinhibit natin yung ibang gram negative at yung ibang gram positive - Bakit naging differential? Kasi merong lactose. Kaya niyang idifferentiateyung lactose fermenter, mula sa mga non lactose fermenter. - Kung may carbohydrates, lagging may pH indicator. Neutral red pa rin ang pH indicator. Bakit need ang pH indicator?kasi kapag naferment ang lactose, merong acid. Kapag may acid, dark pink, kapag wala, colorless. - Ang sabi ditto kapag H2S positive, bakit black? Sabi niyo po kanina ang H2S ay colorless. kaya nga meron tayong indicator, other than the indicator meron tayong sulphur source kaya siya nagiging black 6. BISMUTH SULFITE (BSA) AGAR - Sulphur source is sodium thiosulfate and H2S indicator is a peri ammonium citrate.  Selective: - Ano ang magpoproduce dito ng mga dark color? Syempre sila For: Salmonella spp. (Salm. typhi has distinct appearance) lactose fermenter. Like Escherichia, enterobacter and even si Inhibitors: Bismuth Sulfite CHO Incorporated: Glucose Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 ****Salmonella typhi colonies appear as black colonies with silver - As you can see here, dahil phenol red ang ginamit mo, kapag may metallic sheen acid na naform upon mannitol fermentation there will be yellow colonies and that is staphylococcus aureus. - Usually is selective to salmonella, specifically the salmonella typhi - Eh bakit hindi tumutubo yung ibang ogranisms? Kasi nga po merong (typhi is a sub specie, salmonella is the genera) 7.5 sodium chloride, ayaw ng karamihang bacteria ang salt but - When we write subspecie, kung hindi isinulat yung specie, the specie stapyholoccus can tolerate that. is interica. Salmonella interica typhi. Pag hindi sa nomenclature, pag hindi isinulat kasama ng genera yung specie at nilagay mo yung sub specie, yung subspecie hindi dapat naka underline or italic. 7. BRILLIANT GREEN (BGA) AGAR  Selective:  For: Salmonella spp. Except for Salmonella typhi (nor underlined or italic.  Inhibitors: brilliant green 10. LOWENSTEIN JENSEN (LJ) MEDIUM  CHO Incorporated: lactose  Original Color – light green Salmonella spp. colonies appear as white colonies resembling a  Selective: snowflake surrounded by brilliant red medium For: Mycobacterium spp. Inhibitors: Malachite Green (inhibitor of respiratory tract normal flora)  SPUTUM SAMPLE needs to be: 8. THIOSULFATE CITRATE BILE SALTS SUCROSE 1. Decongested/Digested – to dissolve the thick (TCBS) AGAR mucus/mucin that might betrapping the bacteria in the sample. N-acetyl-L-cysteine (NALC) is usually used.  Original Color – light green/olive green 2. Decontaminated – to eliminate normal flora that  Selective: contaminates the sample. For: Vibrio spp. Inhibitors: thiosulfate, citrate, bile salts for gram positive NaOH is usually used. and other gram negative bacteria. SELECTIVE MEDIUM FOR Neisseria spp. – usually composed  Differential: sucrose INDICATORS bromthymol blue (BTB) of chocolate agar base with antibiotics - Pag carbohydrate (sucrose) may partner yan na pH indicator (bromthymol blue) - Before you culture microbacterium, kailangan itong magunder go into decongested and decontaminated. - So marami tayong gingaamit na decongestant/ digesting agents para madissolve yung mucus or mucin na pwwede magtrap sa bacteria, example don is si N-acetyl-L cysteine (NALC) at ang partner na decontaminant is si sodium hydroxide. 9. MANNITOL SALT (MSA) AGAR - Upon incubation, makikita na kapag ka meron na halimbawa iniincubate with light. Usually mga tutubo is photochromogren  Original Color – light/salmon pink - Scrotochromogen tawag sa mga organisms or micro bacterium  Selective: na Pinatubo without light pero may pigment pa rin. \ For: Staphyloccocus spp. - Ang microbacteria ay may 2 groups, one is photochromogen, Inhibitors: high concentration of salt (7.5%) they have pigment kapag inincubate sila with light. Two is  Differential: scrotochomagen with pigment and light. Meron din non INDICATORS: phenol red photochromagen, they do not reduce pigment, with/ without - Btw ang NSA, meron siyang laman na high salt concentration (7.5) so light. Negative ang pigment. Positive man or negative yung kaya ito itolerate ng staph at si staphylococcus kaya nya iferment si light. Pang apat is yung mga rapid growers kasi yung 1-3 natinn mannitol. Kaya kapag nakakakita tayo ng yellow colonies… why na groups, this are slow growers, and 4th classification, is rapid yellow? Poag phenol red, pag may naproduce na acid, ang kulay growers. niyan yellow, pag wala naman, red or dark pink. Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 For Neisseria, as you can see, each culuture medium, may iba iba - Methyl Red (MR) – used for the detection of bacterial silang gram positive inhibitors, gram negative, fungal, and even pathogen that metabolize glucose using the MIXED ACID proteus. Ang proteus nagsaswarm/ swarming. Halimbawa, super PATHWAY calm ng tubig taapos ng drop ka ng water, parang mag concentric - Voges-Proskauer (VP) used for the detection of bacterial waves. Ganon ang swarming. So contamination siya ng pr0teus. pathogen that metabolizes glucose using the BUTYLENE GLYCOL PATHWAY Ano naman kung may swarming, maooverwhelmed ng swarming - Simmon Citrate Agar (SCA) – used for the detection of yung morphology ng Neisseria and even baka hindi rin tumubo si bacterial pathogen that can utilize citrate as a sole source of Neisseria. Might as well lagyan ng inhibitor for proteus. Which is carbon usually, trimethoprim. Walang trimethoprim ang Thayer martin. Kapag nagpositive si citrate, dapat may growth tsapos possibility is may green parin siya, possible rin pag naging blue or utilization ng citrate. - Triple Sugar Iron Agar (TSI) – used for the determination of bacterial pathogen’s ability to ferment glucose(1 part), sucrose(10 part), or lactose(10 part). It can CULTURE MEDIA FOR ANTIBIOTIC SUSCEPTIBILITY/SENSITIVITY also detect sulfide production and gas production. TESTING (AST) - Lysine Iron Agar (LIA) - used for the determination of bacterial pathogen’s ability to decarboxylate or deaminate #6 step lysine. It can also detect glucose fermentation, sulfide production and gas production. - Ditto magdedetermine kung ano yung tunnel ng antibiotic na - Moeller’s Broth – used to detect lysine decarboxylation, magiginh capablemagihnhibit. May dalawang reaction: resistant ornithine decarboxylation, and arginine dihydrolysis or susceptible. - Stuart’s Urea Broth or Christensen Urea Agar – - Resistant- not capable of inhibiting bacteria used to detect bacterial pathogen that hydrolyze urea substrate - Susceptible- capable of inhibiting bacteria - Kadalsan kapag susceptible ang reaction, eto yung binibigay na antibiotic sa mga patients. - Most bacteria:  Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB)  Kapag agar/plated, there is presence of colonies if lumalaki siya of the medium.  Kapag broth, may makikitang turbidity, means positive ang growth. - Haemophilus spp.  Mueller Hinton with Chocolate Agar Base or Haemophilus Test Medium (HTM) Agar - Mycobacterium spp.  Middlebrook 7H10 or 7H11 Medium CHARACTERISTIC/BIOCHEMICAL CULTURE MEDIA - Step#5 - Sulfide Indole Motility Medium (SIM) - used for observation of hydrogen sulfide gas production, indole production, and motility. - Btw for motility, diba semi solid yan, so usually parang naka butt and orientation. - Kapag ng inoculate tayo ng organism, hanggnag gitna lang. usually ang ginagamit ay inoculating needle. Magtatransfer ng organism, hanggang sa gitna ay iincubate ng 37 degree celcius or 18-24 hours. Kinabuksan ang makikita diyan kapag positive ang motility, merong makikitang turbidity/haziness around the line of stab Downloaded by Clandestine Sy ([email protected])  lOMoARcPSD|12161157 CLINICAL BACTERIOLOGY BACTERIAL CULTIVATION WEEK 4 Downloaded by Clandestine Sy ([email protected])