in chapter 34 we're going to talk about clinical microbiology and immunology and so in this chapter we're going to look at the field of clinical micro and um it's used to detect and identify pathogens that are ideological agents of infectious disease we're going to look at the identification and how it's based on results of morphological physiological biochemical and iminological procedures and time may be critical in life-threatening situations so rapid indication systems and computers can also be used to help speed up the process so this chapter closes with um in vitro antigen antibbody interactions and um particularly those that are used in diagnostic tests so in this first section we're really just looking at that overview of some of the terms that we're going to see in here so a clinical microbiologist is a microbiologist that um their main function is to isolate and identify microorganisms from clinical specimens and to do this as quickly as possible so they're going to perform antimicrobial um susceptibility tests to the organisms um their concern again is to find out what is the um pathogen a clinical specimen this is human material that's tested it's examined um there has to be um special uh careful skill in handling the specimen so you're not contaminating it um and this material is used to stud uh used to determine the presence or absence of a particular organism so this um schematic is showing you the patient specimen is evaluated in different techniques so this is figure 34.1 and um you can see that um rapid tests are used uh bacteria and fungi will go through biochemical identification bacteria fungi protozoa and viruses can go through eliza assays um multipplex systems complement fixation those types of assays microscopy where we're looking at uh organisms in the light microscope um viruses can be seen in the electron microscope cultures um how to purify them and amplify them and look at the cell pathology of viruses um how does that virus impact the cell uh biochemical test this is used for identification and sensitivity so you know what uh sugar are they using are they producing oxygen um that type of thing and then molecular testing uh looking at the nucleic acids sequencing uh toxin detection in a variety of microbes so standard uh practices that we have um and these are things that we've talked about in class uh you need to make sure that you're washing your hands um before you work with any of the lab materials and after um eating and drinking in the lab is not a good idea don't do it um handling your eyes your face you know putting food um you know having any food laying out mouth pipe heading is a big no no um there's a lot of policies that are used to handle sharps and you know in our class we put um any of the um any of the trying to get this box moved uh any of our slides um or sharp broken glass that's that is contaminated always needs to go into a biohazard a sharps container um and then of course uh making sure you're careful not to splash or having aerosols things of that sort you always want to make sure that you disinfect your table before and after your work um that's really important cultures um any material even though it may not be pathogenic um in lab here you need to treat everything as a BSL2 uh just to make sure that you're you know protected from you know anything that could potentially be infectious um the biohazard symbol u make sure that you recognize that symbol and know where to um dispose of materials whether you're here in our lab or if you're in a clinic make sure you know how to handle that material uh the CDC has come up with VSSL levels and again you need to be comfortable with um what you know what do you do if you're exposed um of course here in the classroom let me know if you're in a hospital setting you need to let your infectious control uh people know is typically um but of course your trainers your supervisors um let them know make sure that you report any injury in the lab as soon as possible um just because you know in case there is a problem so when you think about a risk group um this is really not known to be a consistent um cause of disease in a healthy individual so uh lactobacillus uh fibrio the um organisms so the risk group um associated with human disease may be a potential hazard if it's uh a percutaneous injury or it's been ingested salmonila a sharikia coli the 0157H7 staff indigenous or exotic agents with a potential for aerosol transmission um your cineia herpes virus uh the herpes virus uh coxelia so some of these organisms can be uh dangerous these um dangerous and exotic agents that have a high risk for life-threatening disease um Ebola is a big one smallox uh hemorrhagic fever um those types of things so there are some organisms that would fall in a risk group um again uh when you think about uh when you think about the risk group um what individual could be exposed to that um material so looking at the biosafety levels again um when you're studying in this chapter um go back and look at the biosafety levels in our uh lab manual as well so these again were designed by the CDC and um the point of the biosafety levels is to help classify and identify organisms on how to handle them so um you know make sure that you know um your precautions things that you need to do um to um keep yourself safe so biosafety level one this is standard practice there's really no barriers required um like PPE lab coats gloves eye protection as needed so that's you know on on your decision uh for a VSSL1 um the bench and and sink of course are necessary again this is uh BSL 2 so it's everything of BSL 1 plus um limited lab access that's the reason you don't get to come in here in our lab and work without uh me or um a lab assistant you know making sure that you're comforter comfortable with the biohazard signs sharps containers um you know be comfortable with what to do with waste products you know throw things in the biohazard bags um things of that sort and always make sure that you are following up with biohazard safety um policy in your clinic wherever you're working hospital doctor's office um urgent care whatever um bio safety cabinets these are uh to contain uh devices so um that's the reason we have microbes stored um we have uh the chemicals and the reagents that you use stored away a BSL2 you should of course be wearing your lab coat gloves are a good idea face and eye protection um of course as needed um but definitely uh lab coat uh BSL1 facilities again um for the BSL 2 plus an autoclave and we do have an autoclave your BSL 3 this is your BSL 2 plus um this is controlled lab access all waste is dec uh decontaminated which all of our waste is as well here but in a BSL3 lab co lab clothing decontaminated before laundering is a must and there is a baseline serum test done so the cabinets or other physical containment devices are used um again in a BSL3 um protective lab clothing gloves and respiratory protection is needed um in some of these facilities what you're working with so BSL2 facilities plus a self-closing double door negative air pressure air exhaust not recirculation and um that air is recirculated away from uh the hallways bsl 4 um this is BSL 3 plus so really you're using different clothes to go in and out um you shower when you exit and you decontaminate all material when you exit class three glove box uh so this is um the cabinet based work or a class 2 laminer flow hood the cabinets are with full body positive pressure suits um so these are your your top um agents infective agents uh BSL3 facilities plus a separate building or zone um dedicated heat ventilation air conditioning vacuum and decontamination system so this is um a BSL4 when they're working with those organism it's almost like its own lab a separate unit or a separate place so when you're trying to identify an organism from um an unknown so you don't know what the organism is so a specimen should be representative of the diseased area and there must be adequate amount of the specimen so that you can run different diagnostic tests the quality of course needs to be there make sure that the quality is there of the specimen you don't have uh a lot of debris in with the specimen um there's no contamination um you've got the microorganisms that are indigenous to the skin and uh the mucous membranes make sure that you have got as clean a catch if you're doing a urine sample or as clean a sample as possible making sure that microorganisms that are normally there are not contaminating that specimen that you're trying to analyze um it needs to be passed on to the clinical lab properly um so make sure it's marked you can see that here make sure that it's in a bag and it's marked properly and then [Music] um obtained prior to the administration of any antimicrobials um you want to be able to test the sample and um you're making sure what type of treatment to follow up with so this is a laminer flow hood and you can see the grate here so this is to um recirculate air so that um this interior portion is um sterile and no contaminate no contaminants that could possibly come out again the hood that we use in this class is not a laminer flow hood um but these are very um these are very important in a clinical setting so when you're going through identification of these organisms you will use microscopy um you will use growth and biochemical tests uh you can also use rapid methods of identification if you're looking at a bacteria phase you can type it um we can use molecular methods and analyses um to look at metabolic function so there's preliminary identification material uh or um ways to identify the microbe so remember in uh lab 6 and 8 we looked at growth um and that I think was over in our identification section exercise 35 you can see what a colony looks like make sure you're paying attention to the colors is their colors remember Sudamonus has a green color seratia Marcusens has a red color so um uh sudamonus arro um arugganosa the green but um you get these different um characteristics that you need to be able to identify so a culturebased method uh ways that we look at the cultures uh most of your bacteria and fungi you can find pure cultures um so that you can you know take a look at that um the growth and how it looks um you can also use imunomagnetic beads to help in um analyzing small small particles that have antibbody around them so if an antibbody is targeting a specific microbe you can use these imunomagnetic beads to bind to that uh complex so when it's mixed with a clinical sample the antibbody is going to bind to the pathogen binding of the pathogen to the beads so that magnet is going to help to pull the beads out of solution and then you can inoculate the culture there's additional biochemical tests and we'll use these um I guess by the time you hear this we'll probably already have done it but uh in your unknown identification so table 34.4 four has different tests and many of these you're going to use in identifying your unknown because that's what we have to do to make sure that we have the correct um organism there's dichomous keys that are available uh the Bergies manual is a very important uh text that we use it's exercise 39 in your lab manual and it will help you to uh do a flowchart of what organism do you potentially have so this is showing you a dichomous key for some of the important um organisms that that we have that we use in class so um these are for gram positive bacteria and you'll see here that um so this is why you do a grand stain first and you're checking for the pepidoglycan and the functional form of the pepidoglycan molecule is in the paraplasmic space of the cell wall of bacteria of the proarotic cell so you have coxus and basillus shape and then you go through the flowchart and you look to see what um you can say yes or no to so let's just look at a couple so let's say that you have a grandpositive coxus shape so does it grow in air um yes it'll take oxygen to grow and then you can look and test for catalase so um if it grows in air it should produce catalase and then from catalase you're looking at glucose fermentation or not if it ferments glucose then we'll keep going further if it does not then it is a streptocous and then you can look at bile solution and go on down uh if it is a plus it'd be streptococcus pneumonia if it's not then we would go with bile esculine and then test that so that's a streptococcus group D and so on so you can see with this key you can have your unknown and then by doing different tests with your organism it can help pinpoint down to what organism you actually have this is the chart for gram negative bacteria same thing we're looking at the shape so we're doing the gram stain first we're getting the shape is it a coxus is it a basillus and then we go on through um the um sequence so it's a gram negative basillus grows in air look at this canamy that's one of the drugs that we used so if it does not um if kamy doesn't impact it if it's resistant if it's resistant here's a couple of the microbes it could be if it's susceptible here's the microbe the fusa bacterium that it could be what if it grows in air and um uh we test with oxidase oxidase glucose negative or glucose positive and then look at where you would take it from there so um glucose negative then um if it's fermented it would be an intereroacteria family which is a huge family if it is oxidized then you would know um again your organisms as you go so this is a very helpful chart to um help identify what organism you have what bacteria you have um so again by the time you have probably used the API 20E um this is a uh quicker way to to see multiple tests so larger labs are going to use these um and the reason is that you can test all of these different reactions with one test so again it's it's really important um to realize that you know you're trying to uh you're trying to find the uh uh organism what organism you have so that you uh so that you can treat the patient for um whatever is going on for them so this would be uh the API 20E system and this is only going to be for the interobacteria family so there's 20 different inoculation tests in here um the results you convert to a seven or a 9-digit profile number and then you compare that profile number to the index the profile index from the company and then you can identify your organism so when you're trying to identify fungi um you can use direct microscopic examination u looking at with using fluorescent dyes you can examine cultures you can do cerological tests for uh antifungal antibodies uh yeast they can be identified with a rapid ID method so several different ways to uh test for fungi when we think about micro oscopy um this is direct examination of specimen or examination of specimen after uh different staining procedures are performed um there's different staining procedures for bacteria fungi protists so you can see all of this under um microscopy the hybrid um produce a monoconal antibbody specific for a single epitope so these can be fluorescently labeled and used for a rapid accurate culture confirmation uh imofllororesence stains um these can stain specimen with fluorescent dyes that will emit visible colored light so there's direct imofllororesence and there's indirect imofllororesence direct imofllororesence after the fixation to a slide the specimen is stained with a fluorescently labeled antibbody and then um that um is directed at the cell surface antigen an indirect imofllororesence after a known antigen is attached to a slide then the patient's serum which would be the anti- serum is applied if the antibodies specific for the antigen are present then these are detected with a fluorescently labeled secondary antibbody so a couple of ways to uh identify those through microscopy these are some of the um figure or uh pictures to show you what the uh direct and indirect imofllororesence looks like so this is figure 34.5 a is showing you the direct fluorescent and B is showing you the indirect so your unknown antigen the antibbody labeled with a fluorescent die uh once they form the imino complex then uh you can uh see these it would be they were there would be visible visible fluoresence in that then you've got a known antigen and you've got your uh patient serum and then your unknown or your known antibbody serum so the known is going to bind because that's you know it's going to do that and then uh you'll get the fluoresence after you add the uh second antibbody that's fluorescently labeled and then it'll bind to the uh antigen and then of course if um that happens with your patient sample then you know your patient has antibodies to this pathogen if there's no binding um if there's no antibodies in the serum and your patient serum has been added you add the fluorescent antibbody they're not going to bind to the other antibodies uh your antibbody too so you're not going to have any fluorescent so this would be a negative uh result when you think about molecular methods of how micro or how we identify organisms this is um looking at anabolic processes and products so some of these procedures we've already talked about um like protein comparisons enzyme characterizations uh nucleic acidbased composition um and this again is in your uh text in your reading um nucleic acid hybridization nucleic acid sequencing so really analyzing the nucleic acid nucleic acid based detection methods are also used so singlestranded DNA molecules have been cloned from the organism or prepared by PCR and then they're used in hybridization procedures your ribosal RNA genes can be sequenced to identify bacterial strains so this is called ribotyping um also there's GLA gas liquid chromatography that can be used um these are going to help identify specific microbial metabolites uh cellular fatty acids um usually for non-polar substances that can be extracted in ether plasmid fingerprinting um so separation and detection of the number the molecular weight the restriction pattern of different plasmids and um PCR this is um really used a lot um in the develop um since PCR has been developed so this is allowing um um us to or u in identification to make multiple copies of the DNA and um analyze that to match it up so our known DNA to our unknown DNA so that we can see um what organism we do have so PCR results here this is showing you units of fluoresence and then the cycle because you can do multiple cycles with PCRs so Aven flu A um H5 plus control is in the black line and you can follow it then Aven flu a H5 plus stimulated samples so this is the blue shades so kind of a a medium blue a darker blue and a light blue so it's these three colors that we're seeing then we have a negative control and a negative sample so our negative control is a little bit uh darker that's kind of hard to see in this uh picture um and then our negative sample so this is the negative sample this kind of lighter gray and then uh the darker I guess medium gray is that negative control but it's kind of hard to follow those in here anyway you wouldn't expect the uh aven flu to be in the negative control and the negative sample you would expect it to be in the positive um control and then these are the samples that were extracted and um analyzed so these three are uh avian flu A and you can know that because they follow along with the um units of fluoresence in this figure so you can deduct that it is uh a blue so ribot typing again uh the gene that um most used to identify the bacteria in blood and other cultures is the 16S rna gene so looking at this and uh you want to go through sequencing of course so uh amplifying it with PCR and then sequencing that and so so when we think about um cerology so um serotyping uh this is looking at in clinical settings you want to uh detect if um the antigen and antibbody is visible um detecting antigen and antibbody can of course be very important in diag diagnosing the condition of your patient and to be able to interpret the data so serotyping is antigen antibbody specificity that's used to differentiate various strains or serars of an organism found in serum samples so the presence of IGM indicates a recent active infection while IGG is evidence of a past or a chronic infection or even vaccination aglutination uh these are visible clumps or aggregates of cells or of coated latex microsphheres if red blood cells are aglutinated the reaction is called hemoglutination um there's a few different tests that are used the widle test is direct aglutination test this has been used to detect uh typhoid fever latex aglutination is used in pregnancy tests to diagnose the uh the micro the micotic um helmet or bacterial infections viral hemoglutination um inhibition tests are used to diagnose flu or other viral infections and a glutination test can be used to measure antibbody tighter so this reciprocal of the greatest dilutions showing a glutination reaction so viral um hemoglutination this figure in chapter uh 34 this is figure 34.7 so this is showing you the red blood cells measles virus and then the hemoglutination uh this is red blood cells with measal viruses and then the anti-measal antibodies with your patient so if the measles virus um or then the measles virus is neutralized with the antibodies um and you have this um um neutralization so you do not have hemoglutination to occur here like you do here so it lets you know if the uh person has had that virus before tube glutenation uh this is an example to look for tighters uh so the tighter in this example is 160 so right here is the 160 and so um there's no aglutination in the next tube the next series so we do not have a glutenation here so the tighter of this um example would be 160 complement fixation uh complement is uh used to detect the presence of serum antibodies to a pathogen uh it's currently used to diagnose um some viral infections fungal infections um protozoal diseases so in um this example here uh this is figure uh 34.9 and what you're seeing here is you have your test serum so we have the antigen that we're adding to uh the solution and we have antigen and antibbody added so we're forming immune complexes um here we have the complement the free compliment and then um more complement that's going to fix the immune complexes here and then the indicator cell is um liced so it has a negative test result the indicator cell here is an anti- urethroite antibbody so the cell is not liced and it shows a positive test so this is a way to test with um compliment fixation liza assays these are enzyme linked amunosorbent assay so these are going to link enzymes to antibodies uh double antibbody sandwich assay so this is going to detect the antigens in a sample and there's indirect uh iminosorbent assay uh so with the direct test wells of the microtrer are coated with antibodies specific to the antigen the test sample is placed in the antigen um so here we're showing um indirect Eliza here and direct Eliza here so again antibbody is added to the well the test sample is added um if it has the antigen then the antigen uh will be retained in the well after it's been washed out uh the second antibbody is then added here's the second um antibbody uh it's conjugated to an enzyme and it's specific to the antigen the second antibbody will be retained in the well after washing if the antigen was retained in the previous step then there is a substrate added um then that reaction is going to occur if the conjugated enzyme is present in the well it produces a colored product like you can see here this this yellow color and you can see them in this 96 well plate this is an Eliza assay plate well it's a 96 well plate it's used for other things but it's used in Eliza assays so your indirect method this is going to detect serum antibbody so the whale of the microtrer plate is coated with the antigen specific to the antibbody test serum is added if the antibodies are present they're going to bind to the antigen and be retained after washing an antibbody is going to be added um against the test imogloabbulin and the second antibbody is conjugated to the enzyme and is only going to retain in the well after washing if the test antibbody is present so here is here's the antibbody initially and then the whale is rinsed here's the second antibbody that comes in um and so again the the antibbody will remain the conjugate will remain after washing if the test antibbody was present you're going to add substrate again and if the enzyme is added the reaction only occurs if the conjugated antibbody uh so the test antibbody was present in the well the colored product of this reaction is detected through spectr photometry okay so a couple ways with Eliza assays then we have the um lateral flow assay uh this figure that you're seeing is um figure 34.11 and so lateral flow assays are um that you have your uh viral antigen that's in the specimen so you add this to your um assay um sample you've got your labeled antiviral antibbody here so they're going to bind to the virus and then they're going to migrate they're going to uh proceed down the filter paper as they're going here's the test line and the control line when that liquid sample comes to the test line if the antibbody is captured um at the test line uh the antibbody well the antibody will be captured that has the virus there the ones that do not did not bind are going to go to the control line so you'll see a few at the control line so the unlabeled antiviral antibbody are here on the test line and the anti-IGG are here so this can help to um tell if there is an antigen antibbody complex that's been captured if the patient lacks the antigen only the control line will be visible so if your patient is positive it's going to look like this if your patient is negative it's going to look like this western blotting imunolotting um this is where proteins are separated by electrofaresis uh they're blotted on nitroc cellulose paper and or sheets and then they're treated with a solution that contains the enzyme tagged antibodies there's also imuno precipitation uh soluble antigens form an insoluble immune complex that can be detected and you can see that here um this is figure uh 34.12 and what you're noticing here is you have antibbody added you have the antigen added so the zone of antibbody excess you've got antibbody and antigen present then you have a large lattice aggregation um so this uh combination this combination of um antibbody antigen and then the zone of antigen excess where there's more antigen so what you're able to see here is uh the preip ring where you have antibbody bound to the antigen here you have the antibodies um at the bottom and antigen at the top so you get this um optimal ratio of antigen and antibbody right here in the middle that preip imbunion diffusion uh this figure is figure 34.13 and uh this involves precipitation of immune complexes in an augur gel and so you can see this auger gel gel uh these wells have been cut out and um so you have a single radial immuno diffusion assay to quantify you've got a double diffusion assay or the um octalone um technique that's used these are going these lines of preip form where the antibodies and antigens have diffused and they met this really determines where the antigens share identical determinants so you've got your uh your control antibbody here you've got your antigen in the uh center test serum one and test serum 2 so what you're doing you've got the known antigen and you would expect to see the precipitation bands here the precipit bands here because the control antibbody is here and the antigen is here with test serum number one there's no U band so this is negative to the antigen uh this test serum 2 does have a band so it is positive to the serum and this is just a side view to show you how you would add that flowcytometry and fluoresence these are going to detect single and multiple microorganisms on the basis of cytotric parameters so you're going to be able to see the fuorochromes with these um you can go back to chapter 33 um we didn't cover that chapter but you can look at it um if you've got interest in flowcytometry a lot of your uh well your hematologists are going to use it your um uh oncologists are going to use it depending on um the type of um cancer that they're looking at for oncology but the flowcytometer is going to force a cell through a laser beam and then that light um the scattered light will be uh counted or measured and the fluoresence as the cell passes through the beam is what it's picking up cells can be tagged with the fluorescent antibbody um directed against the specific surface antigen so it can really um be um valuable for identifying um pathogens so that takes care of chapter 34 let me know if you have any questions