Microbiology lect.16 5/04/011 Specimen collection and handling in the laboratory.

This is basically chapter 13 from your textbook. So the proper diagnosis of infectious disease requires the following: (this is obviously, for the physicians)
y Taking a complete patient history. y Conducting a thorough physical examination of the patient y Carefully evaluating the patient's signs and symptoms y Implementing the proper selection, collection,transport, and

processing of appropriate clinical specimens In base of all these ,the physician will suspect a certain disease or a certain number of diseases and based on these he will request a few clinical samples to be taken from the patient and sent to the microbiology laboratory. Slide 4 : This is a typical flow chart of steps in diagnosis of the infectious disease, so patient with symptoms of an infectious disease consult with clinician, clinician makes preliminary diagnosis and write order for the laboratory tests. Appropriate specimens are collected from the patient and transported to the microbiology laboratory . In the lab. the preliminary process the sample and might obtain certain preliminary findings and can report this delivery findings to the physician and then will continue working on the sample and perform a thorough investigation .So they can do various types of diagnostics methods, they can remove bacterial cultures or fungal cultures,they can do gram stain and other tests in order to identify the pathogen .Also they might do something called "ANTIMICROBIAL SUSCEPTIBILITY TESTING ",in order to know which type of antimicrobial agent is effective against the pathogen and then they send all their finding to the physician ,so the physician within used

1|Pa ge

preliminary finding or the final findings in order to reach the final diagnosis and initiate the proper therapy. So basically any type of sample uptake from a patient is called CLINICAL SPECIMEN ,and the clinical microbiology lab can process various types of clinical specimens. So anything you can think of, that come from patient is considered a clinical specimen. For example, blood, bone marrow, bronchial washings, sputum, CSF, cervical and vaginal swabs, feces, hair and nail clippings, pus, skin scrapings, synovial fluid, throat swabs, tissue specimens, urethral discharge material, urine, and urogenital secretions.; all these are considered clinical specimens and all of these can be processed in the clinical microbiology lab to determine whether contain any pathogen. And in order for the lab to reach a proper diagnosis( an accurate diagnosis) the specimen needs to be of a very high quality; we will talk about the various characteristics that constitute or give the sample high quality.
" Need to be a very clear communication between the physician and

the health care workers and the laboratory technicians in order to properly obtain the sample and in order to properly analyze and process the sample.
" Also if the patient is highly infectious and you should treat the entire

sample that's highly infectious there should be care in transporting and processing the sample in order not to infect ourselves while working with the sample and also in order not to contaminate the sample with our own normal flora. The clinical microbiology lab will give the sample and eventually tell the physician;for example ,if you find a Salmonella Typhea in the sample, that doesn't mean necessarily that the lab is telling the physician the patient has salmonellosis diagnosis, they say only that they found this organism or this virus or this fungus in the sample .The ultimate diagnosis is on the

2|Pa ge

physician himself. So the physician will take all the information about the clinical signs and symptoms ,patient history and will reach to the diagnosis. So the lab. Helps him in reaching diagnosis but they don't make the diagnosis themselves. So we have 3 important components regarding specimen's quality: The specimen needs to be properly selective ,for example, someone having a skin abscess obviously the proper sample will be an abscess aspirate ,not a throat swab. So you take the sample from a relevant site. Proper specimen collection basically, is the same thing ,if you have a wound on your hand ,you shouldn't take a swab from that wound ;The best sample is by using an aspirate,using a needle and a syringe ;and also all samples should be properly transported to the laboratory ;so usually transport, the best transport is the fastest transport and the one that doesn't really lead the sample to spoil. So the person collecting the clinical specimen is not acting on his own, so there's a certain patterns ,rules and guidelines that he is following and these rules are guidelines on how we obtain the sample ,from where we obtain the sample and how to transport the sample ;all these were actually stepped before handling, by the clinical microbiology lab. So the clinical microbiology lab .usually has certain book ,for example Laboratory Policies and Procedures Manual, that contain in highly detailed way how to obtain a high quality sample and how to transport it . So the person who collects the specimen is automatically responsible for its quality; well experienced professionals usually will give you higher quality than who have just started this type of job. Here are some general guidelines that ensure that you are having a good quality sample, we have already talked about them
y Specimens must be properly selected. y Specimens must be collected properly, using proper tools.

3|Pa ge

y Material (specimens) should be collected from a site where the

suspected pathogen is most likely to be found. So if you have Asepthecymia most likely you are going to have a blood sample not a skin scrape.
y Specimens should be obtained before antimicrobial therapy, if

possible. Usually if a patient start antimicrobial therapy and you take a sample from that patient there will be very little chance to obtain ,( or to isolate) the pathogen or might not get the pathogen at all .
y The acute stage of the disease is the most appropriate time to

collect a specimen. The acute stage responds to the highest number of pathogens within your body; so that's the best time to take the sample.
y Specimen collection should be performed with care to avoid harming

the patient.
y A sufficient quantity of the specimen must be obtained to provide

enough material for all required diagnostic tests. For example if you have Asepthemcymia ,you should take a good amount of blood ,maybe 10-2 ml of blood from the patient ,because you want that blood to do multiple tests so 1ml will not be enough to do all analyzing tests.
y All specimens should be placed or collected into a sterile container

to prevent contamination.
y Specimens should be protected from heat and cold and promptly

delivered to the laboratory. Many pathogens are highly sensitive to low temperature or high temperature so we should keep the sample temperature as close as possible to the room temperature.
y Hazardous specimens must be handled with even greater care to

avoid contamination of couriers, patients, and healthcare professionals. Samples of highly infectious materials, such as samples taken from someone with tuberculosis, SARS, HIV or hepatitis viruses, so these should be handled with extreme care.

4|Pa ge

y Whenever possible, a sterile, disposable specimen container should

be used.
y The specimen container must be properly labeled and accompanied

by an appropriate request slip with adequate instructions. Whenever you send a sample to the lab. Make sure that you properly label it with the patient name, date ,time of collection and much information about the patient as possible. So the lab. More carefully will handle the specimen and also knowing that certain organisms are suspected to be present will help the lab. In doing proper diagnosis by doing extra test that might help in the identification of the microorganism.
y Specimens should be collected and delivered to the lab as early in

the day as possible to allow sufficient processing time. If the lab finishes or closes at 5 pm and you send them a sample at 4 pm there's a very little chance that they are going to do anything on the sample that day. So the earlier in the day you send it the more likely that they are going to process the sample that day. Waiting is just a waste of time and the sample might spoil over night. Specimen transport: Most labs. Are situated in same location or very close to the physician;so you will not really have to deal with this most of the time ,but if you are sending a sample to a remote location or to a reference lab then you have to properly pack the sample and properly label the sample in order to reach the destination safely. Slide 11: the picture : This is your sample, a tube contain some bacteria ; what they did here is they added some water proof tape to make sure that nothing will spell outside the tube ,then they surrounded the tube with absorbent packing material ,to protect it from any fall and even if breaks this absorbent material will absorb all the liquid ,so when you open

5|Pa ge

it will not splash on your face .They place the whole thing into two containers ,one small and one bigger container ,and they properly labeled the outside of the larger container .So they added a bio-hazardous label to tell the people receiving the sample this is potentially bio-hazardous and obviously they are sending it to a distant location ,they add the address for delivery. Now certain types of specimens that are typically handled by clinical microbiology laboratory:  Blood is a very common clinical laboratory specimen. Blood in healthy people is usually sterile; if you find bacteria inside the blood then this is referred to as bacteremia; so bacteremia means blood containing bacteria. This is in contrast with something called Septicemia ,which is basically bacteria in blood that is actively multiplying and producing various types of toxins and septicemia is associated with clinical symptoms such as chills and fever .So septicemia is a life threatening condition whereas bacteremia might not necessarily be life threatening. If you brush your teeth, just by the process of brushing your teeth, some bacteria from your mouth will end up in your circulation so you might have a transient bacteremia after brushing your teeth or cleaning your teeth, you don't develop disease , but septicemia is a very lighter disease. A major problem in collecting blood specimens, is protecting the blood sample from contamination from skin normal flora; so how do you obtain the blood? Usually you use a needle ,goes from the skin to the vein ,as it's going through the skin there a chance that this needle is contaminated with skin normal flora .To prevent this , or to minimize the risk ,usually you take the sample using an aseptic technique ;so basically the typical site for obtaining a blood sample is in the arm ,in a place called the

6|Pa ge

"anticupidal fossa" and the vein that you usually extract blood from is the Anticupidal vein ;so this is the site of extraction ,in order to prepare the site you have to use some kind of disinfectant ,such as 70% alcohol and you rub this area in a circular motion ,so basically you are drawing away bacteria from the central site to the outside. If you do back and forth ,the bacteria will spread and brought back to the central site. Then you let the alcohol dry and then you obtain the venous blood sample . This will minimize or prevent the contamination of the blood sample with skin normal flora.  Urine :is another common clinical sample . In normal humans, normal urine inside the bladder, before it goes to the outside, normally is sterile .So if you obtain a sample from the bladder and you culture it you will not find any organisms. However a problem with urine is the outside of the urethra is usually contaminated with bacteria derived from the skin, so even though the urine is normally sterile it will get contaminated while urinating. If you do a culture for urine usually you will get some organisms in the urine. So in order to minimize the amount of bacteria in urine when you do a urine culture, you have to perform or obtain a sample called a cleancatch, midstream(CCMS) urine. Obviously with urinary tract infections the bacteria will be multiplying in the bladder and urine will no longer be sterile, but you need to discrete between normal contaminated urine and truly contaminated urine . In order to obtain clean-catch, midstream(CCMS) urine ,you have to instruct the patient to properly collect the sample ;firstly you tell them how to clean the outside of the urethra with soap and water and tell them to discard the first amount of urine ,so the urethra or outside the urethra is usually contaminated with some bacteria so when they avoid the first amount of urine ,they are washing away as much bacteria as possible from the urethra; then you tell them after they avoid the first

7|Pa ge

part of urine, collect the middle part of the urine ; usually they fill half of the container and sealing without touching the inside of the container and send it to the lab. To be processed. This is called clean-catch because you clean the urethra ,midstream(CCMS) because you discard the first part of the urine and obtain the middle part, which is supposedly the least contaminated with skin normal flora. If you culture a sample of urine obtained using this method ,you will get some bacterial colonies on a blood agar plate ;using this method you will not completely eliminate contamination of the urine , you just reduce it ; so the number of colonies here can be multiplied by a certain number and obtain something called "NUMBER OF COLONY FORMING UNIT per ML OF URINE " . Normal urine from a healthy person without a urinary tract infection will contain less than 100 000 CFU per ml or urine, whereas people with urinary tract infection will usually have 100 000 and more CFU per ml urine; 100 000 is the limit between healthy and infected. What we usually do ,once obtained the clean catch midstream urine is ,you take a sterile loop ; the loop ,when you put it in urine and take out will carry a small amount of urine on it ;you take this amount of urine and spread it evenly on the surface of blood agar plate then incubate this plate at 37 degrees for all the night and count the number of colonies .We have two types of loop that can be used : 1. One loop caries 0.01 ml of urine 2. Smaller type that carry 0.001 ml of urine We like more to use the smaller one ,so we usually take this small amount of urine and spread it on a plate and observe how many colonies are present in that volume .Any number you obtain on the plate ,you multiply it by the DILUTION FACTOR; 1 ml will have 1000 time more bacteria than you obtained from this loop, you multiply by 1000 if you are using 0.001 ml and if use the 0.01 ml you multiply by 100. Let's assume you counted 90 colonies on the plate, and u you used the 0.001 ml, then you have in

8|Pa ge

that urine 90 000 colonies forming unite per ml. this is interpreted as normal urine, don't have urine infection. If you obtain 150 colonies and you used the smaller loop, then you have 150 000 cfu per ml urine, which indicates the urine is infected, urinary tract infection. More than 100 000 is infected , less is normal urine . Once you do this kind of urine culture , you determine this is high number of bacteria ,the next step is try to obtain the bacteria in pure culture and identify the bacteria using a variety of methods. After that, you propone something called ANTIMICROBIAL SUSCEPTIBILITY TESTING, to determine which type of antimicrobial agent is effective in killing that bacterium and finally report the final findings and results. Understand well the concept of bacterial count ,because I might give you a question in the exam requiring some calculations.  Another common sample is Cerebrospinal Fluid (CSF): The physician will require a sample to be taken if a patient has meningitis , Encephalitis or Meningoencephalitis ; i. ii. iii. Meningitis is inflammation or infection of the membranes (meninges) that surround the brain and spinal column. Encephalitis is inflammation or infection of the brain. Meningoencephalitis is inflammation or infection of both the brain and meninges. Obtaining a cerebrospinal fluid sample is to be done only by the physician ; and done using an aseptic techniques . CFS samples are considered to be emergency or stat specimen in the lab because of  The first ,many of the pathogens present in a CFS sample are very sensitive ,so they might die if not processed immediately

9|Pa ge 

Patient with meningitis ,or encephalitis or meningoencephalitis are usually in very critical conditions ;this is a life threatening situation ,so you have to process the sample very quickly .  Sputum . Sputum is pus that accumulates deep within the lungs of a patient with pneumonia, tuberculosis, or other lower respiratory infection. The problem with sputum is that when you ask a patient to provide you sputum ,they don't know how to do the sputum ,most likely might have some sputum and a lot of saliva ,and saliva is not helpful for the lab. it doesn't contain the pathogen which is present in the lung ,in case of lower respiratory tract infection . An example of a case where you have to ask for a sputum sample is Tuberculosis. Better specimens can be obtain by bronchial aspiration or transtracheal aspiration .  Throat Swabs Are indicated when you have an upper respiratory tract infection and depending of the suspected disease,the way you collect the sample will differ slightly ;we just say : that we usually use a cotton swab and you try to touch the inflamed area or postural area on the back of your throat ,your tonsils and take out the swab without touching the tongue or any part of the mouth.  Wound specimen Are best taken using an aspirate needle and syringe, you inject deep in the wound and try to take the pus deep in the wound;if you take a swab ,usually the swab will only obtain the material on the surface and the surface material will be contaminated with other skin normal flora; so you will get mixed types of bacteria from the skin surface or from a surface swab.  Fecal Specimens

10 | P a g e

Ideally, fecal (stool) specimens should be collected at the laboratory and processed immediately to prevent a decrease in temperature, which would allow the pH to drop and cause the death of many Shigella and Salmonella species. We have few pathogens associated with Gastrointestinal tract infections ,but one of the most important are Shigella and Salmonella ;these organisms are highly sensitive to low Ph ,acidic Ph; if you allow this tool sample too down ,the Ph will go down and we kill this Salmonella and shigella and make it very difficult for the lab to obtain or to isolate these pathogens. You either have to obtain the sample and process immediately or if cannot be processed immediately the person who obtained the sample can add a certain type of preservative to the stool sample which will prevent the Ph from falling. When the lab obtains the fecal sample they can do a variety of tests to it, first they can observe microscopic characteristics of the stool ,what color has ,whether is diarrheic or solid or semisolid and then they can do microscopical examinations or stool culture. Also in the case of parasites they can look for the presence of any types of infective stages or diagnostics stages of protozoa and molds (I'm not sure about molds types of ova or eggs release by helminthes . The clinical microbiology lab is part of a bigger department called the Pathological department; slide 23 shows the organization of pathology department . Within the clinical microbiology lab we have different sections : 1. Bacteriology 2. Mycology 3. Parasitology 4. Virology 5. Mycobacteriology 6. Immunology The main responsibilities of microbiology lab. Are the following : it was not clear ) ;they can look for trophozoids and protozoa and variant

11 | P a g e

-Obviously they receive the sample from the physician and then they process this sample in various ways .One of the first thing they usually do is try to observe the sample macroscopically with naked eye,to see if the sample is bloody or if it contains pus or if it has any characteristic (like odors).; then they usually do a direct wet mount, gram stain for the sample and from there they can do other tests such as culture and biochemical identification of the pathogens. When a lab. Reaches or identifies a pathogen we call this "speciation" ; speciation is giving a name to the pathogen present in the sample. Once the lab. Reaches this they can perform the antimicrobial susceptibility test whenever is appropriate to do it. Some examples of function of each of the microbiology section: Bacteriology : is concerned with identifying bacterial causes of disease and usually do antimicrobial susceptibility test ,as a usual routine ;the way that the lab personal in the bacteriology section identify the bacteria and give it a name ,is by identifying certain characteristics that are unique to the various species. For example some bacteria are Gram positive whereas others are Gram negative, cell shape, whether the bacteria is motile or not, presence and location of spores and presence or absence of various enzymes and so on In the lab, once you go to the practical session in the last two labs, there are a lot of biochemical tests that you can do that help you in identifying the bacteria. Basically, clinical microbiology lab. People are crime serial investigators, like CSI, they collect clues from the sample itself microscopically and macroscopically and biochemically and eventually from all the clues they take it they will reach the proper identification .Nowadays things are moving from the manual test to automation or semi-automation ;there are new systems that help the bacteriology people in reaching the proper identification of bacteria. One example is API-20E for identification of Enterobacteriaceae . So basically once you obtain bacteria from the pure

12 | P a g e

culture ,you inoculate various containers on this strip ,you incubate the strip for a short period of time or maybe overnight ,these swells will change color and depending on the color pattern you get you can identify the bacteria . Another example of the criteria that helps you in the identification of bacteria is Hemolysis pattern.Some bacteria can produce enzymes that completely destroy RBCs and if you grow these bacteria on a blood agar plate ,this bacteria will grow in large colonies and destroy RBC around them and this is called B-Hemolysis ,which indicates complete distruction of RBCs ; some bacteria can produce enzymes that partially degrade RBCs and usually partially degradation will lead to a greenish coloring of the media ,this is called -hemolysis . Some bacteria cannot destroy RBCs and they will not change the color or appearance of blood agar plate, these are called gamma hemolysis . Mycology section is for diagnosis of fungal infection (mycoses) ;the samples arriving to the mycology section are the same as those obtained by the bacteriology section ,except that they also have hair and nail clippings and skin scrapings, because many of the fungal diseases are present on the skin and nails and hairs. We have a variety of procedures that can be used to identify the fungi for example we have KOH preps, biochemical tests (for yeasts), and a combination of microscopic and macroscopic observations (for moulds). Slide 32 is an example of a mold colony ,this is for Aspergillus fumigates , common cause of Pulmonary Infections in Immunosuppressed Patients. Slide 34 is an example of Penicillium Species; you can see different types of organisms will lead to different properties for their colonies. Parasitology section , They allow for the identification and diagnosis of parasitic diseases ;parasites can be unicellular such as Protozoa ,so you can look for certain stages and trophozoid stages of protozoa in stool specimens or other

13 | P a g e

clinical specimens ; some parasites can be multicellular such as worms ,you can look for worms in stool specimen or look for their eggs. Virology section : Help in diagnosis of viral infection and viruses can be diagnosed using a variety of methods including immunodiagnostic tests, cytological or histological examination, electron microscopy, molecular techniques, virus Mycobacterial section also called the TB Lab. : Assists clinicians in the diagnosis of tuberculosis (TB).Various types of specimens are submitted, but sputum is the most common type. Mycobacterium spp. are identified by the acid-fast staining procedure and by using a combination of growth characteristics (e.g., growth rate, colony pigmentation, and morphology) and a variety of biochemical tests.

Done by: Sara Ibdiwi

14 | P a g e

Sign up to vote on this title
UsefulNot useful

Master Your Semester with Scribd & The New York Times

Special offer for students: Only $4.99/month.

Master Your Semester with a Special Offer from Scribd & The New York Times

Cancel anytime.