Clinical Laboratory Procedures PDF

Dr. Radhwan Asal Clinical laboratory Cultures and sensitivity Culture is defined as a laboratory test by which samples from body specimens are cultivated in a special growth medium in order to isolate the microorganisms that may be present. Culture is a highly effective laboratory method for identi...

Dr. Radhwan Asal Clinical laboratory Cultures and sensitivity Culture is defined as a laboratory test by which samples from body specimens are cultivated in a special growth medium in order to isolate the microorganisms that may be present. Culture is a highly effective laboratory method for identifying the microorganisms that cause infectious disease and for obtaining a definitive diagnosis. Why it’s done? Generally, culture and sensitivity test is done to: Detect and identify bacteria that may be causing an infection Identify the best antibiotic to treat the infection. Culture samples Specimen collection for culture and sensitivity testing must be carried out under standard precautionary techniques in order to avoid contamination of the specimen. 1. Urine sample A urine culture may be done when an abnormal result from a urinalysis (such as an increased number of white blood cells) indicates an infection. Sampling Patient must not take any antibiotics before testing by 48 hours. Specimens should be obtained in a sterile containers and must be of sufficient quantity Morning mid-stream specimen of urine for quantitative bacteriuria examination is ideal specimen. The sample must be in the lab for testing within 30 minutes after taking. Note: Using diuretics or drinking a large amount of liquid. This may dilute your urine and lower the concentration of bacteria, causing inaccurate test results. Page 1 of 10 Dr. Radhwan Asal 2. Stool sample A stool culture is done to detect and identify certain types of bacteria that can cause symptoms of an intestinal disease which include prolonged diarrhoea, bloody diarrhoea, and an increased amount of gas, abdominal pain and fever. Note: More than 50 different kinds of bacteria normally live in the intestines. However, disease can result if numbers of abnormal bacteria in the intestines increase. Sampling Patient must not take any antibiotics before testing by 48 hours. Specimens should be obtained in a sterile containers and must be of sufficient quantity. The sample must be in the lab for testing within 30 minutes after taking. 3. Wound sample A skin or wound culture is a test to detect and identify bacteria that may be infecting the skin or a wounds such as injuries, burn, surgical wound or animal bites. Note: symptoms of an infection often include pain at the site, redness, tenderness, swelling, and warmth, red streaks toward the body, swollen lymph nodes, and the presence of pus. Sampling Patient must not take any antibiotics before testing by 48 hours. Specimens should be obtained by a sterile swab and must be of sufficient quantity. Pus sample must be taken from deep inside the wound. 4. Blood sample A blood culture can detect and identify bacterial infection in the blood. The blood does not normally contain any bacteria but bacteria can enter the bloodstream as a severe complication of infections (like pneumonia, osteomyelitis or meningitis), during surgery (especially when involving mucous membranes such as the gastrointestinal tract), or due to catheters and other foreign bodies entering the arteries or veins (including intravenous drug abuse). A bacterial infection is usually serious because the blood can spread the bacteria to any part of the body. Page 2 of 10 Dr. Radhwan Asal Note: A blood culture is also done to evaluate unexplained fever, (Pyrexia of unknown origin (PUO)). Note: Bacteremia is a medical term referring the presence of bacteria in the blood while Septicemia is a medical term referring to the presence of pathogenic organisms in the blood-stream, leading to sepsis (in which multiplying bacteria release toxins into the blood stream and trigger the production of cytokines, causing fever, chills, toxicity, and reduced blood pressure). Sampling Incubate media bottles before taking the sample at 37 degree for at least10 minutes Put the label on the bottle indicating the patient identification Disinfect the skin carefully to avoid contamination with skin microorganisms. Blood is obtained by inserting a needle into the vein then draw about 10 ml of blood and put it into two culture bottles containing broth to grow aerobic and anaerobic microorganisms. Inoculate the culture bottles carefully and incubate the bottles in incubator Do not store inoculated bottles in the refrigerator Culture media Types of Culture media 1. Nutrient agar: Is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. Agar is a polysaccharide formed of agarose and agaropectin , it is used to solidify culture media because: It has a highly gelling capacity Free of substances toxic to bacteria Note: This medium is the base of different types of media. Page 3 of 10 Dr. Radhwan Asal 2. Blood agar plate (BAP) Contains mammalian blood (usually sheep or horse), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. 3. 3. Chocolate agar (CHOC) A type of blood agar plate in which the blood cells have been lysed by heating the cells to 56 °C. Chocolate agar is used for growing fastidious bacteria. 4. 4. MacConkey agar (MAC) A selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. 5. 5.Salmonella Shigella Agar (S.S Agar) It is a selective growth medium used in the isolation of Salmonella and Shigella species from clinical samples. Preparation of growth media Weight an accurate amount of solid agar media Add distilled water to the solid media and dissolve well Dissolve the ingredients by using glass Boil the mixture in autoclave Preparation of culture plates Cool the media to about 50 C Pour the media into sterilized Petri dish Allow the media to cool and solidify keep Petri dishes in an inverted position during storage in refrigerator to prevent condensation of moisture on the plate Page 4 of 10 Dr. Radhwan Asal Cultivation of bacteria How to inoculate a plate: 1. Heat the loop and after cooling it in alcohol transfer a loop-full of culture on the plate 2. Spread bacteria on the solid surface of the plate as in the shape to obtain a single colony 3. The Petri dish is kept in inverted position 4. Re sterilize the loop again 5. Inoculated media should be incubated at 37 degree as soon as possible Page 5 of 10 Dr. Radhwan Asal Gram stain Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. Staining mechanism Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains purple while Gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. 1. Gram-negative bacteria Are those bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counter stain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. Examples  Gram-negative cocci  Neisseria gonorrhoeae  Gram-negative bacilli  Helicobacter pylori  Vibrio cholera  Brucella Page 6 of 10 Dr. Radhwan Asal  Salmonella 2. Gram-positive bacteria Are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counter stain and appearing red or pink. Examples  Gram-positive cocci  Staphylococci  Streptococci  Gram-positive bacilli  Bacillus anthracis Gram stain procedure: Prepare a clear slide and put one drop of normal saline Take specimen from the colony and mix it well with normal saline. Leave for dryness on air then fix it on burner. Then pour Crystal Violet stain on the slide and leave it for 1 minute. Wash gently with water. Then pour Iodine stain on the slide and leave it for 1 minute. Wash gently with water. Wash with Alcohol to decolorize the slide. Then pour counter stain (fuchsine) on the slide and leave it for 1 minute. Results under microscope: Violet cells refer to Gram Positive bacteria (G+ve) and red cells which stained by fuchsine refer to Gram Negative bacteria (G-ve). Page 7 of 10 Dr. Radhwan Asal Antibiotics Sensitivity Test Once the microorganism has been cultured and identified, it is tested for sensitivity to specific antibiotics. Sensitivity testing completes the process known as culture and sensitivity. Purpose Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. Principle Testing for antibiotic sensitivity is often done by the Kirby-Bauer method (Agar diffusion method). It is the most commonly used sensitivity test and involves the inoculation of a special agar plate with the organism to be tested. Once the plate has been inoculated, antibiotic disks are placed onto the surface of the agar and the plate is turned upside down and incubated overnight. The reaction between the antibiotic disks and the organism creates a zone of inhibition, an area of no growth, around each antibiotic disk if the antibiotic is effective against that bacterium. The size of this zone is used to classify the organism as sensitive (S), meaning the antibiotic inhibits growth (the organism is sensitive to the antibiotic); resistant (R), meaning the antibiotic does not inhibit growth (the organism is resistant to the antibiotic); and intermediate (I), Page 8 of 10 Dr. Radhwan Asal meaning the antibiotic inhibits growth somewhat, but not enough to be effective. Procedures Streak plate method 1. Plates are dried by opening the plate in the incubator for 10-20 minutes 2. Carefully picking one colony by the sterile loop 3. Then it is streaked several times along one edge of a Petri dish containing nutrient agar. Note: Swab the surface of the agar completely (you do not want to leave any unswabbed agar areas at all). 4. After completely swabbing the plate, turn it 90 degrees and repeat the swabbing process. 5. Allow the surface to dry for about 5 minutes before placing antibiotic disks on the agar. 6. Just quickly pick up the disc and move it to the appropriate place with the sterile forceps. 7. Lightly touch each disc with your sterile inoculating loop to make sure that it is in good contact with the agar surface. 8. Incubate for 37 degree for 24 hours. Page 9 of 10 Dr. Radhwan Asal Interpretation: Place the metric ruler across the zone of inhibition, at the widest diameter, and measure from one edge of the zone to the other edge. Zone diameter is reported as S (sensitive), R (resistant), or I (intermediate). *** Good Luck *** Page 10 of 10