Microbiology, Lecture 17

8/7/2019 Microbiology, Lecture 17

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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 patienty Carefully evaluating the patient's signs and symptomsy 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 thelaboratory 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 SUSCEPTIBILITYTESTING ",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


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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 andthe health care workers and the laboratory technicians in order to

properly obtain the sample and in order to properly analyze andprocess the sample.

" Also if the patient is highly infectious and you should treat the entiresample 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


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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 weobtain 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.


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y Material (specimens) should be collected from a site where thesuspected 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, ifpossible. 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 tocollect 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 harmingthe patient.

y A sufficient quantity of the specimen must be obtained to provideenough 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 testsso 1ml will not be enoughto do allanalyzing tests.

y All specimens should be placed or collected into a sterile containerto prevent contamination.

y Specimens should be protected from heat and cold and promptlydelivered 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 toavoid 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.


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y Whenever possible, a sterile, disposable specimen container shouldbe used.

y The specimen container must be properly labeled and accompaniedby 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 inthe 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


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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 asbacteremia; 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


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"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 aurine culture, you have to perform or obtain a sample called a clean-

catch, 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


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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 COLONYFORMINGUNIT 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 andspread 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 urine2.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


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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 SUSCEPTIBILITYTESTING, 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.

Meningitis is inflammation or infection of the membranes (meninges)that surround the brain and spinal column.

ii. Encephalitis is inflammation or infection of the brain.iii. 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 verysensitive ,so they might die if not processed immediately


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Patient with meningitis ,or encephalitis or meningoencephalitis areusually 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 SwabsAre 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 tryto 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 specimenAre 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


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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 moldsit

was not clear ) ;they can look for trophozoids and protozoa and varianttypes 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.Bacteriology2.Mycology3.Parasitology4.Virology5.Mycobacteriology6.Immunology

The main responsibilities of microbiology lab. Are the following :


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-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 ofdisease 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 andlocation 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


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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

ofRBCs ; 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) ;thesamples 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.W

e have avariety 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 ofPulmonary Infections in Immunosuppressed Patients.

Slide 34 is an example ofPenicillium 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


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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

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Microbiology, Lecture 17