Graduation presentation in practicum subject part 1

Graduation presentation in practicum subject Instruction name: Dr. doaa houdari Done by: Nagham Zaiden Hiba seif al Deen Noor Othman Fatima Abdullah.

Laboratory Department……. 1 Phlebotomy 2 Bacteriology 3 Biochemistry 4 Hematology 5 Serology 6 Parasitology 7 Histology and Cytology 8 Blood bank.

Phlebotomy.

What is phlebotomy? Phlebotomy describes the act of removing blood from a patient using a needle. This can be for the purposes of laboratory testing as a diagnostic tool to narrow down a differential or can be used therapeutically for certain conditions. What are the different types of phlebotomy? There are two main types of phlebotomy: Venipuncture and capillary puncture. 1-Venipuncture is the most common type of phlebotomy. It involves taking blood from a vein, usually in the arm. 2-Capillary puncture is less common than venipuncture, but it can be used when veins are not accessible or if a large amount of blood is needed. It involves taking blood from a small vessel in the skin, such as a finger or toe..

Equipment:.

Procedure:.

What is the tube that is used in phlebotomy ?.

What type of specimen is collected in lab? 1-(Blood, Plasma, and Serum)Cells (e.g., RBC, WBC, etc.): 2-Tissues: 3-Urine: 4-Saliva: 5-Feces/stool:.

6-Semen 7-Bone Marrow 9-Body fluid 8-pus ( (vaginal, pleural, CSF….).

When specimen is rejected in lab ? 1-The information on the requisition does not match the information on the specimen label. If the patient name or source does not match, the specimen should be collected again. 2-The specimen is not submitted in the appropriate transport container or the container is leaking. The quantity of the specimen is inadequate to perform all tests requested. 3-The specimen transport time is more than 2 hours and the specimen has not been preserved. An anaerobic culture is requested on a specimen in which anaerobes are indigenous. 4-Specimen is dried up. And One swab was submitted with multiple requests for various organisms 5-More than one specimen from the same source was submitted from the same patient on the same day; blood cultures are an exception. .6-Gram stain of expectorated sputum reveals less than 25 white blood cells (WBCs) and more than 10 epithelial cells per low power field and mixed bacterial flora..

Bacteriology.

Key test for gram (-) bacteria: •Oxidase test: is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain in aerobic respiration. Organisms which contain cytochrome c are oxidase-positive and turn the reagent blue/purple. Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless, and are oxidase-negative. Result:  Gram-negative bacilli Enterobacteriaceae are oxidase- negative  Whereas many other bacilli such as Pseudomonas are positive..

Citrate: When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to blue above pH 7.6. Results: 1.E.coli: citrate negative (green) 2. Klebsiella, Citrobacte and Entrerobacter are citrate positive (blue)..

•Indole: Indole is one of the metabolic degradation products of the amino acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole, pyruvic acid, and ammonia. The indole test is based on the formation of a red complex when indole reacts with the active chemical in Kovac reagent. Results: Klebsiella oxytoxa + Klebsiella pneumonia and enterobacter -.

Urease hydrolyzes the urea into ammonia, water, and carbon dioxide.The presence of the enzyme is determined by inoculating an organism to broth or agar that contains urea as the primary carbon source and detecting the production of ammonia. Results: Motility test:(ability to move) Result: 1.A positive motility test is indicated by a turbid area extending away from the line of inoculation. 2.A negative test is indicated by growth along the inoculation line but no further. E.coli and Salmonella are positive . Shigella and Klebsiella are negative. • Urease test: Proteus + (red) E.coli - (yellow).

Klingler test :• KIA contains only two carbohydrates: glucose and lactose. It is used to differentiate Enterobacteriaceae based on the ability to ferment carbohydrates and produce hydrogen sulfide (H2S). -Bacteria that ferment any of the three sugars in the medium will produce acids which will change the color from red to yellow. -If hydrogen sulfide (H2S) is formed during growth, a gray or black streak of iron sulfide is seen. -If gaz is produced, bubbles or cracks are seen in the bottom..

K/K K/A A/A + GAS A/A+H2S K/A +GAS + H2S pseudomonas shigella Ecoli citerobacter salmonella.

LIA test: is used to determine whether a Gram negative rod decarboxylates or deaminates Lysine and forms Hydrogen sulfide (H2S). -When Glucose is fermented, the butt of the medium becomes acidic (yellow). -If the organism produces Lysine decarboxylase, Cadaverine is formed, Cadaverine neutralizes the organic acids formed by Glucose fermentation and the butt of the medium reverts to the alkaline state (purple). -if the decarboxylase is not present, the butt remains acidic. incubate the tube at 37ºC for 18 to 24 hours.

Control K/A R/A K/K K/K+H2S Citrobacter Protus E.coli Salmonella.

Key test for gram (+) bacteria? Catalase test: This enzyme catalyzes the release of water and oxygen from hydrogen peroxide. (H2O2+ catalase = H2O + O2). The rapid production of bubbles when bacterial growth is mixed with a hydrogen peroxide solution is interpreted as a positive test. Staphylococcus + Streptococcus and enterococcus - Results:.

Coagulase test : Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin. It is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (Negative.

DNase test : The test is used to determine the ability of an organism to produce the enzyme DNase with hydrolyzes DNA into oligonucleotides. If no indicator, the oligonucleotides are soluble in 1M HCl and forming a clearing zone around the bacterial growth but DNA are insoluble. After incubation for 24 hours; •Flood the surface of agar with 1N HCL solution. •Tip off the excess acid. •Allow the reagent to absorb into the plate. •Observe for clear zone around the colonies within 5 minutes. Results: S.aureus + CNS -.

CAMP test: Certain organisms (Streptococcus agalactiae or group B streptococci) produce a diffusible extracellular protein (CAMP factor) that acts synergistically with the beta-lysis of Staphylococcus aureus to cause enhanced lysis of red blood cells. A: beta-hemolytic Group B strep (arrow shape) B: other beta-hemolytic C: S.aureus  This test is used also to identify Listeria monocytogenes, which is a CAMP positive organism..

Bile esculin test: This test is based on the ability of certain bacteria, notably Enterococcus species and group D streptococci, to hydrolyze esculin in the presence of bile. Hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin. Esculetin reacts with ferric ions to form a black diffusible complex. Salt tolerance: To differentiate between Enterococcus and group D streptococcus salt tolerance test (6.5% NaCl) is done. Bile esculine NaCl enterococ cus + + Group D + -.

MSA test: This tests for the bacteria’s ability to tolerate 7% salt concentration and ferment mannitol. It is selective because it selects for salt tolerant bacteria. Reading Results: 1.If the organism is tolerant to salt it will grow. 2.If the organism is not tolerant to salt it will not grow. 3.If the salt tolerant organism can ferment mannitol then there will be yellow zones around the colonies. 4.If the salt tolerant organism cannot ferment mannitol then the media will remain pink..

Type of culture media MacConkey: selective for gram (-) bacteria. The presence of crystal violet inhibit the growth of gram (+) bacteria. Its differential between lactose and non-lactose fermenting organism. The decrease in PH after fermentation of lactose on lactic acid is the cause of the color change into pink. Lactose fermenting: 1. E Coli: • citrate (-) • gram (-) rod • Most member of the family of Enterobacteriaceae • Indole (+) • Yellow colonies on CLED agar • Oxidase (-).

2. Klebsiella (mucoid): • citrate (+) • gram (-) bacilli • nosocomial infection • urease (+) • cause septicemia, pneumonia • catalase (+) • A/A +gas Non-lactose fermenting: (yellow) 1. Proteus: gram (–) rod • swarming on BA • chocolate odor • Highly motile + urease (+). Klebsiella oxytoca Indole + Klebsiella pneumonia Indole- Proteus vulgaris Indole + Proteus mirabilus Indole -.

2. Pseudomonas: gram (-) bacilli • oxidase (+) • catalase (+), citrate (+) • fruity odor • green color + give Mueller Hinton green color • flower shape + grow on 42 degree • cause of nosocomial infection 3. Serratia: • dark pink color • flower shape (cause of nosocomial infection). 4. Morganella. On the plate of non-lactose fermenting there is 5% E.coli. (Citrate should be negative). CNA (contain blood): selective for gram (+) bacteria. The presence of colistin and nalidixic acid (antibiotics) inhibit the growth of gram (-) bacteria. It’s differential based on the type of hemolysis...

Beta hemolysis Complete hemolysis (yellow) Alpha hemolysis Partial hemolysis (green) Gamma hemolysis Lack of hemolysis Mucoid species on CNA agar = gram (-) which have resistance on colistin..

Gram stain Tests S OR R Staph Cluster Catalase and DNase + Sensitive on lysostaphin + resistant on bacitracin Micrococcus Cluster DNase - Resistant on lysostaphin + sensitive on bacitracin Strep gram(+) cocci Chain Catalase - Bile salt – Sensitive on bacitracin Enterococcus Chain Catalase - Bile salt + Vancomycin resistant S.aureus Novobiocin sensitive S.saprophitics Novobiocin resistant.

Streptococcus 1.Alpha hemolysis:  streptococcus pneumonia (gram + diplococcic):  green colonies  donuts shape on the plate.  Optochine sensitive  (+) bile solubility test (turbid after the lysis of the bacteria by the action of Na deoxycholate)  Disease caused by pneumonia: meningitis, otitis media, snottiest, pneumonia. 2.Beta hemolysis: -group A (pyogenes) present in the upper respiratory tract  bacitracin sensitive  catalase (-)  PYR + (same for enterococcus)..

-Group B (agalactia) present in the genital tract of the female • neonatal meningitis  positive camp test. (Arrow) • Don’t grow on MAC agar. 3. Gamma hemolysis: -Group D: bile (+) salt (-) enterococcus: bile (+) salt (+) (Bile esculin test is + for both).

•SS agar: used for the detection of salmonella and shigella (stool). Contains bile salt and brilliant green (inhibition of gram +bacteria), lactose as a sugar and neutral red as a PH indicator. Na thiosulfate and ferric acid for the visualization of H2S. (Proteus, salmonella and Citrobacter give H2S) 1.Salmonella: ferment the sugar and give a pink color with a black center.(H2S) • Urease (-) • indole (-) • flagellated, facultative anaerobic bacilli • non-lactose fermenting 2.Shigella: • color less. If there is presence of H2S we need to replate: we put some isolated colonies on selenite (enrichment broth for stool) then we replate on MAC, BA and SS agar to confirm the presence of salmonella + some sugar like lysine, urea and kligler are used for detection..

•Chocolate agar: enriched agar for the growth of gram (+) & (-) bacteria. Contain X (hemin) and V (NAD) factor. Bacteria that grow on CHOC but not BA: homophiles influenza and Neisseria species. • Blood agar: For gram (+), gram(-) and fungi..

•Thayer marten Agar: selective for N.gonorrhae (gram (-) diplococci like coffee beans). VCNT- agar: 1.Vancomycin: inhibition of gram (+). 2.Colistin: inhibition of gram (–). 3. Nestatin: inhibition of fungi. 4.Trimethoprim: inhibition of proteus. •TCBS and APW (incubation for 5hr) Selective for vibrio cholera (yellow colonies). Vibrio cholera ferment sucrose and have gamma shape under microscope..

Gram Stain • Gram (-): pink = thin peptidoglycan. • Gram (+): purple = thick peptidoglycan...

Acid Fast Stain 1-Gram (+) bacilli: nocardia which is filamentous. Weakly acid fast stain 2-Mycobacterium tuberculosis (lepra). Procedure: 1-Primary stain: carbon fushin (red) which contain phenol that stimulate the penetration in the lipid layer of the mycobacterium. 2-Heating for 5min (fixation) 3-Acid alcohol (mycobacterium keep red color). 4-Methyl blue (other bacteria appear blue). Result: Blue background and red bacilli..

Olive shape with purple color. Germ tube: to identify the type of yeast present. -It contains serum of any patient + yeast from the culture media then we incubate for 3 hours. Sensitivity=1.8-2.2.

Blood bottle: Used for the detection of bacteremia and septicemia. Contains SPS which is an anti-coagulant and inhibit phagocytosis. Adult (40ml) = 2 aerobic and 2 anaerobic. Baby (3ml) -We put the bottle in the machine for 5 days if no alert then no bacteria so we record as no growth. -If the alert was given we get the bottle out of the machine and we take blood of it using needle and we subculture it on MAC, BA and CHOC agar + gram stain + direct sensitivity antibiotics. The pathogens that are frequently identified in blood culture: S.aureus, E.coli, some members of the family Enterobacteriaceae, enterococcus species, pseudomonas aeruginosa and candida albican..

Antibiogram: Antibiotic sensitivity testing or antibiotic susceptibility testing is the measurement of the susceptibility of bacteria to antibiotics. It is used because bacteria may have resistance to some antibiotics. Performed on Mueller Hinton agar. Sensitivity: mix isolated bacterial colonies in normal saline precisely. sensitivity= 0.5-0.63 1.For staph: -Oxacillin + ciphoxidin to identify MSSA and MRSA organism. -Clindamycin + erythromycin (D test): if there is a D shape surrounding clind. Then both antibiotics are resistant even one of them was sensitive..

2.For G.N (LF/NLF): ESBL arc: presence of cloud shape (synergy) between cephtazidin(CAZ) + amoxyclovanic acid(AMC) + CRO The presence of synergy indicate the high % of resistance on several antibiotics. (VRI organism) ERT: it’s okay if sensitive Resistance > double check: ERT + emypenime + meropenem + colistin +tigecycline. If resistant: 1-Ecoli = CRE 2-Klebsiella = KPC 3-Pseudomonas = MRD Mueller Hinton blood: Moraxella, pneumonia and streptococcus..

API: The analytical profile index, or API, is a classification of bacteria based on biochemical tests, allowing fast identification. (This API-20E test strip is used to identify the enteric gram negative rods (although API makes a variety of other test strips for yeast, Staph, anaerobes). 20 separate test compartments are on the strip, all dehydrated. A bacterial suspension is used to rehydrate each of the wells. If we have NLF organism that we cannot identify and we are out of machines > API because it may be from the family of Enterobacteriaceae. If we have sugar in case of H2S. • Oxidase (+) = non entero (NE) • Oxidase (-) = entero (E).

Urine culture: A urine culture is a lab test to check for bacteria or other germs in a urine sample. It can be used to check for a urinary tract infection in adults and children. The female and male urinary tracts are relatively the same except for the length of the urethra...

We don’t care about gram(+) rod in urine. If we have mixed colonies and the number colonies < 10000 Then the result is (-) . WBC =2-3 > we don’t work on the specimens (number of WBCs should be high) If it is positive count the colonies (CFu/ml) A- ˂10000 CFU/ml are considered normal B- ≥100000 CFU/ml UTI (urinary tract infection) C- from 10000 to 100000 the culture is determinate IN IMAM ALI WE CULTURE THE URINE ON MACONKY(for gram negative), BLOOD(for gram negative and gram positive), SABOUROD (for fungi) AND CHAPNER (LIKE CNA for gram positive)..

VITEK: VITEK is a fully automated system that performs bacterial identification and antibiotic susceptibility testing. (Usually done for patient inside the hospital / nosocomial infection) For the identification of the presence of only one colony of bacteria. Procedure: 1.Numbering tubes based on the patient number 2.Fill the tubes with specific Vitek saline solution all on the same level (sensitivity with isolated colonies) 3.If we are searching for the sensitivity on antibiotics in addition to the ID we take from the mixture in the tube 1 into the second for the same patient= dilution (same for all tubes) 4.Put on the specific cards inside the tubes (LF: 417 / NLF: 419) 5.Scan the cards bar code and place them inside the room 1 of the machine until the alert is given so you can place them in the room 2 6.After the machine finish the process and the results are given tubes are taken for a purity check. (on BA).

Specimens incubation time: CSF and other fluid for 72hours. • CSF: plating on MAC, CHOC and BA. • It’s a sterile specimens = we use sterile loop • 1 drop inside the plate and 5 drops inside the thio (REPLATE) • We should check the number of WBCs: -high n.b of neutrophils = bacterial infection (low glucose) -high n.b of lymphocytes = viral infection Other specimens for 42 hours. 1. Swab (sterile): MAC , BA and thio (enrichment media) If we have a (-) growth we should replate to make sure that results are true...

Sputum: we search for : 1.Strep group A. 2.Moraxella (gram (-) cocci / brown colonies) 3.Pneumonia 4.Staph 5.Yeast Throat: 1.Pneumonia 2.Strep 3.Moraxella (yellow colonies) 4.Staph Moraxella catarales Oxidase and Dnase + Moraxella non- pathogenic DNase -.

Rectal and nasal: DNase(-) -> we don’t need sensitivity DNase (+) -> we need sensitivity (we care about staph) -We perform ESBL -They need incubation for 4 hours. -If patient are coming from other hospital. Semen: we usually found gram (-) bacteria = E.coli Watery stool + blood: -SS agar + selenite +sorbitol -Fermentation of sorbitol: dark pink -Non fermentation: transparent to yellow = E.coli O157.

Brucella : Gram (-) cocci Don’t grow on MAC. It only grow on CHOC and BA. Brucellosis is a bacterial infection that spreads from animals to people. Most commonly, people are infected by eating raw or unpasteurized dairy products. Acinetobacter: Bright pink color Diplococcic appear red Fish odor Gram (-) which have a resistance on several antibiotics (Mult resistant) Sensitive on colistin Cause of nosocomial infection It can cause death.

GARDENELA : it is gram variable bacteria (gram positive and gram negative) that are found in vagina in women and this type of bacteria grow inside agar..