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Unit III Urines • THE URINARY TRACT The Urinary Tract • • Pyelonephritis • Anatomy of the Kidney Cystitis • www.kidneystone911.com Anatomy of the Kidney Capsule: A thin but tough outer membrane that protects the kidney against infection and trauma Renal pelvis: A large funnel for collecting urine from all parts of the kidney, connected to the bladder by the ureter. Medulla: The inner layer of the kidney's interior, which contains triangular structures called renal pyramids. Between pyramids are sections of cortex called renal columns. Cortex: The outer layer of the kidney's interior, about 1 cm (0.4 inch) thick. www.kidneystone911.com The Urinary System • • • • • • • • • • • • • • • • • • • • • • • Urine Terminology: 1. Urinary Tract Infection (UTI): a spectrum of diseases caused by microbial invasion of the genitourinary (GU) tract that extends from the renal cortex of the kidney to the urethral meatus. 2. Bacteriuria: the presence of detectable bacteria in the urine. Patients may be symptomatic or asymptomatic (geriatrics and pregnancy). 3. Coliform: a general term applied to some species of the Family Enterobacteriaceae , including E.coli, Klebsiella and Enterobacter species. Their presence in water (especially that of E.coli) is presumptive evidence of fecal contamination. 4. Pyuria: pus in the urine; evidence of renal disease; a condition in which there are more than the normal number of white blood cells in the urine. >10 WBC’s / HPF 5. Pyelonephritis: inflammation of the kidney and renal pelvis, usually as a result of a bacterial infection that has ascended from the urinary bladder. 6. Enteric: pertaining to the small intestine 7. Costovertebral: pertaining to a rib and a vertebrae 8. Flank: the part of the body between the ribs and the upper border of the ileum. The term also refers loosely to the outer side of the thigh, hip and buttock. • • • • • • • • • • • • • • • • • • • • • • • • • 9. Upper Urinary Tract Infection (U-UTI): A genitourinary tract infection that is limited to the renal parenchyma (pyelonephritis) or the ureters (ureteritis). It is often accompanied by lower urinary tract symptoms in addition to costovertebral (CV) flank pain or tenderness and fever. At times, lower UTI’s precedes the appearance of fever and upper UTI’s by 24 to 48 hours. 10. Lower Urinary Tract Infection (L-UTI): a genitourinary tract infection that is limited to the urethra (urethritis), bladder (cystitis), and in males, the prostate (prostatitis). These infections generally present in adults with dysuria (pain in urination), increase in frequency, urgency and occasionally suprapubic tenderness. 11. Acute Urethral Syndrome (AUS): Patients with this syndrome are primarily young, sexually active women, who experience dysuria, frequency and urgency but yield less than 105 CFU/mL on urine culture. AUS is defined as more than 8 leukocytes per mm3 on microscopic exam of uncentrifuged urine OR approximately 2 to 5 leukocytes/hpf in centrifuged urine sediment. While a good portion of women with these symptoms may actually have a UTI, many have organisms recovered with less than 105 colony counts and the main culprit recovered is Staphylococcus saprophyticus. And still there are others with these same symptoms that have been found to have a STD (sexually transmitted disease) or vaginitis. 12. Prostatitis: a genitourinary infection in males, involving the prostate; fever often present with low back or perineal pain and at times urinary frequency and urgency. 13. Cervicitis: inflammation of the cervix; may occur as an acute or a chronic presentation. Causative agents include sexually transmitted organisms, such a N. gonorrhoeae and C. trachomatis. Symptoms include dysuria, urgency, vaginal discharge and low back pain. URINARY TRACT INFECTIONS • • • Most Urinary Tract Infections are: a. Upper: kidneys, renal pelves, ureters (also called “pyelonephritis”) b. Lower: urinary bladder and urethra (also called “cystitis”) • • • PYELONEPHRITIS: (Upper UTI). Symptoms include: fever, flank pain and thoracic costovertebral angle tenderness *** The same symptoms as cystitis except NO urinary frequency.** • • • • • CYSTITIS: (Lower UTI) - Symptoms include: fever, flank pain, thoracic costo-vertebral angle tenderness AND urinary frequency - Most lower UTI's involve the urinary bladder, with potential spread to the prostate gland in males (prostatitis) and to the urethra in women (Acute Urethral Syndrome) • • • • • • • - the bacterial count considered significant and associated with UTI is >l00,000 CFU/ml the most common cause of UTI's is E. coli. UTI's are the largest cause of nosocomial infections All areas of urinary tract are sterile above the urethra UTI’s more common in females due to short urethra and close proximity to anus UTI’s in men, incidence extremely low until after age 60 and then usually due to prostatic hypertrophy. SYMPTOMS OF KIDNEY DISEASE: • - Fatigue • - Puffy eyes • - Swollen face, hands, abdomen, ankles, feet • - Increased thirst • - Difficult, painful urination • - Foamy urine • - Pink, dark urine (blood in urine) UNTREATED KIDNEY DISEASE CAN LEAD TO: • - Heart disease (including heart attack and stroke) • - Weak bones • - Nerve damage (neuropathy) • - High blood pressure • - Kidney failure (ESRD: End Stage Renal Disease) • - Anemia or low red blood cell count • - Death PREDISPOSING RISK FACTORS: • • • • • • • • • • • • • - Obstruction (kidney stones) - Pregnancy - Age (60 or older) - Obesity - Diabetes (self or family) - High blood pressure (self or family) - Heart disease (self of family) - Race (African American, Native American, Hispanic, Asian, Pacific Islander heritage) - Prolonged use of NSAID’s (Advil, Aleve or Motrin) - Catheterization and/or chronic UTI’s - Autoimmune diseases (i.e., Lupus or others) - Instrumentation - Compromised urine flow DIAGNOSTIC STUDY: a diagnostic study of the urinary tract is indicated: a. after the first infection in males b. in all cases of suspected pyelonephritis c. after the second or third infection in females (within a short period of time) TESTS TO DETERMINE KIDNEY FUNCTION: • MicroAlbumin (detects protein and albumin in urine: < 30) • Creatinine in blood: (0.6 – 1.2 mg/dl) • Glomerular Filtration Rate: (Keep over 90 mls/min) • ** IV pyelogram - can be used to diagnose: • a. anatomical defects • b. loss of renal parenchyma (functional cells) from chronic pyelonephritis • c. obstructive lesions • • • • • • WHAT HEALTHY KIDNEYS DO: - Regulate the body’s fluid levels - Activate Vitamin D to maintain healthy bones - Filter wastes and toxins from the blood - Release the hormone that directs production of red blood cells (erythropoietin) - Release a hormone that regulates blood pressure (aldosterone and ADH: antidiuretic hormone) • - Keeps blood minerals/electrolytes in balance (sodium, potassium, phosphorus, calcium) TYPES OF URINE SPECIMENS : BAP & MAC agars used only. l. CCMS (Clean Catch Mid-Stream) - best all around specimen usually with few skin contaminants. • Allows for maximum recovery for UTI screening. Specimens should be processed immediately • upon arrival. If that is not achievable, then refrigeration is a safe and practical method to hold a • urine until it can be processed. A urine may be refrigerated for up to 24 hours without • significant alteration in bacterial population. However, room temperature for extended periods • of time (> 2 hours after collection) may give erroneous results by showing a definite increase in • the colony count. A "clean-catch" urine sample is performed by collecting the sample of urine in midstream. Men or boys should wipe clean the head of the penis. Women or girls need to wash the area between the lips of the vagina with soapy water and rinse well. A small amount of urine should initially fall into the toilet bowl before it is collected (this clears the urethra of contaminants). Then, in a clean container, catch about 1 to 2 ounces of urine and remove the container from the urine stream. The container is then given to the health care provider. www.copanusa.com 2. CATHETERIZED - should be restricted to those patients who are unable to produce a CCMS and performed with strict supervision and meticulous attention to aseptic technique. Considered sterile. Greater than 10,000 cfu/ml needed before considered significant. (< than 10,000 cfu/ml give descriptive ID only). Refer to flow chart for work up procedure. Foley catheters are flexible (usually latex) tubes that are passed through the urethra during urinary catheterization and into the bladder to drain urine. They are retained by means of a balloon at the tip which is inflated with sterile water. The balloons typically come in two different sizes 5 cc and 30 cc. 3. CYSTOSCOPY - collected in OR by urologist with aid of camera- sterile - should contain no skin contaminants, so all organisms are worked up. Lab may receive urine from several sites that may include bladder, kidney etc. Refer to flow chart for work up procedure. A Cystoscopy is a procedure that is carried out using a specialized endoscopic instrument called a cystoscope. The cystoscope is a long, lighted tube that has a small camera at the end, allowing a view of the inside of your bladder and urethra. A cystoscopy may reveal information that is not obvious on an X-ray. www.gcurology.com • • • • As well as diagnosis, a Cystoscopy may also include treatment for the following: - Removal of bladder tumors - Crushing and removing stones found in the bladder - Removal of obstruction in the bladder - Bleeding in the bladder 4. SUPRAPUBIC URINE - collected through the abdomen into the bladder. Reserved almost exclusively for neonates and small children. Best performed when the bladder is full. It should be treated like a cath urine. Refer to flow chart for work up procedure. www.catheterout.org A Suprapubic (SP) catheter is a tube that enters the urinary bladder through the wall of the abdomen. It may be needed for a variety of reasons. Often, it is used to constantly drain urine from the bladder after extensive or delicate surgery on the genitals or bladder. The suprapubic tube may be connected to a large drainage bag or to a smaller drainage bag that is attached to the leg. 5. BAG URINE - not sterile - a small plastic bag taped to infants for urine collection. Often contaminated with feces. Follow CCMS rules for work up. Remove cover on adhesive tape and press onto genitals. In pediatric population, replace diaper and let child resume normal activities until urine is obtained. www.apexmed.eu UNACCEPTABLE CULTURES: • l. Urine should not be cultured anaerobically a. indigenous flora in urethra and surrounding area b. urinary tract not conducive atmosphere for anaerobic infection • 2. Foley tips - invariably contaminated with urethral organisms • 3. Specimens that have sat at room temperature for more than 2 hours ( colony count) • 4. Specimens that have been centrifuged (concentrated colony count) ORGANISMS CONSIDERED CONTAMINANTS FROM URINE CULTURES • l. Staph epidermidis • 2. Diphtheroids (members of the Genus Corynebacterium) • 3. Gram negative rods ( >100,000 CFU/mL X 3) • 4. Enterococcus species (small amounts) • 5. Lactobacillus • 6. Viridans Strep (alpha strep) • 7. Streptococcus agalactiae (GBS: unless from woman of child bearing age) • 8. Bacillus species CHARLOTTE INSTITUTE OF REHABILITATION: (CIR) Rehab Patients: • • • • • • 1. All organisms from Rehab patients who are paraplegic (paralyzed from the waist down) or quadriplegic (paralyzed from the neck down) get a full work up. Organisms that would normally be considered “contaminants” can become potential pathogens to this type patient. These patients usually have complicated UTI’s due to certain risk factors involved (indwelling urinary catheters or functional abnormalities of the urinary tract) thus making it more difficult to treat and greater morbidity (e.g., kidney damage, bacteremia) and mortality rates. • ** Note: If a patient has any significant degree of paraplegia, he/she will be unable to urinate voluntarily from the moment of the injury. His/Her bladder will fill up slowly and will be full by about midnight on the day of admission. If you leave it, it will overflow, so one should prevent this from happening. The best way of treating him/her is to use regular intermittent sterile catheterization. Infection is rare with this method. It imitates the natural cycle in which the bladder fills and empties. By leaving it almost empty for a significant period, this method relieves the pressure on its walls, both pressure of urine and pressure from the balloon of a Foley catheter. This is important, because distension or pressure of any kind reduces the ability of the bladder to resist infection. The disadvantage of this method is that it requires more nursing care, and if the patient is to do it himself, he must be cooperative. Some consultant surgeons in teaching hospitals say they cannot use this method, because they don’t have the staff, and wonder that it can ever be done in smaller ones. In fact, some smaller hospitals can do it excellently. Don’t use: (1) an indwelling catheter if you can possibly avoid doing so because infection is so common, or, (2) continuous suprapubic drainage, because it produces a small contracted bladder. CULTURE METHODS FOR DETECTION OF BACTERIURIA • • • 1. COLONY COUNT - using a calibrated loop (0.01-ml or 0.001-ml), deliver the inoculum onto the surface of each agar plate streaking in quadrants so that semi- quantitative colony counts can be performed after incubation. 2. POUR PLATE METHOD - most accurate method for measuring degree of bacteruria. Involves dilution with melted agar. Use a Quebec colony counter to enumerate the number of colonies on the plate. • NON-CULTURE METHODS FOR DETECTION OF BACTERIURIA • 1. GRAM STAIN - 1 organism per high power field (HPF) in an uncentrifuged urine reflects a colony count of >100,000 CFU/ml. • 2. GREISS TEST - reagent-impregnated dipsticks designed to detect the presence of nitrites which is indicative of gram negative rods caused by the Enterobacteriaceae. • • • • • • • • • • 3. TTC (triphenyltetrazolium chloride) - reduction of TTC by metabolizing bacteria 4. GLUCOSE OXIDASE - based on metabolism of the small amount of glucose present in normal urine by bacteria 5. AUTOMATED METHODS a. ATP (adenosine triphosphate) - detects bacterial ATP by measuring light emitted by the reaction of luciferin luciferase. Photons are measured correlating the number of bacteria present in sample. b. TURNER system - measures bacteria present before and after short incubation period c. BAC-T-SCREEN - uses filter paper that urine is passed through which holds bacteria and neutrophils. A dye is added and the intensity of the color relates to the number of particles present. Takes about 2 minutes and detects 95% of all positive urines. d. LUMAC - urine screening system employing bioluminescence • e. BACTEC - blood cultures containing tryptic soy agar are inoculated with urine and run on machine to detect growth • f. LEUKOCYTE ESTERASE - an indirect method to estimate the number of segmented neutrophils through the detection of leukocyte esterase activity. A special reagent strip can be used to detect LE in urine. • *** Some of these tests have been considered unsatisfactory because of substantial numbers of false-negative results and sometimes lack of specificity. FAMILY ENTEROBACTERIACEAE • Genera of Family Enterobacteriaceae encountered most often in clinical specimens: • • • • • • • 1. 2. 3. 4. 5. 6. 7. E. coli Klebsiella Enterobacter Hafnia Citrobacter Edwardsiella Proteus • • • • • • • • 1. 2. 3. 4. 5. 6. 7. 8. grouped by DNA relatedness, gram negative rods, aerobic, facultative anaerobes, grow well on routine lab media (especially MacConkey agar) if motile, possesses peri-trichous flagella all nitrate positive all ferments glucose, alkaline/acid and gas all oxidase negative found in intestines of humans and animals, soils and plants drugs of choice for treatment of infections with the Enterobacteriaceae are aminoglycosides, SXT, and third generation cephalosporins 8. 9. 10. 11. 12. 13. Providencia Morganella Serratia Salmonella Shigella Yersina IMViC (Indole, Methyl-Red, Vogues-Proskauer, Citrate) • - used to distinguish between the coliform bacteria ESCHERICHA COLI • • • • the most commonly recovered species A/Ag, IMViC= + + - most strains ferment lactose most common cause of gram negative sepsis and endotoxin-induced shock. Also causes urinary tract infections, pyelitis, pyelonephritis, appendicitis, peritonitis, gallbladder infections, septicemia, meningitis, endocarditis, gastrointestinal disease and nosocomial acquired pneumoniae • • • Can cause enteritis or gastroenteritis syndromes by 4 different mechanisms: ETEC - “traveler’s diarrhea” - caused by an enterotoxigenic strain EPEC - “newborn diarrhea” - caused by an enteropathogenic strain of E. coli that is very invasive and found primarily in infants. EIEC - “enteroinvasive E. coli” - produces an inflammatory diarrhea similar to that caused by Shigella; blood, mucus and leukocytes seen in stool EHEC - “enterohemorraghic E. coli” – E.coli strain serotype 0157:H7 has been associated with serious hemorrhagic colitis, ischemic colitis and hemolytic uremic syndrome characterized by low platelet count, hemolytic anemia and kidney failure. E.coli 0157:H7 produces two cytotoxins (verotoxins I and II). Verotoxin I is a phage-encoded cytotoxin identical to the Shiga toxin (Stx) produced by Shigella dysenteriae type I. This verotoxin shows damage on vero cells (African green monkey kidney cells), hence the term verotoxin. It has been reported that patients with hemorrhagic colitis shed the organism for only a brief period of time; nevertheless, verotoxins may still be detected in the stool (via ELISA method directly from bloody stools that contain the Stx toxin). • • • Continuing with EHEC: • Stool culture for E.coli 0157:H7 may be performed using MacConkey agar containing Sorbitol (MACS) instead of lactose. EHEC does not ferment sorbitol and would appear colorless on MacConkey agar with sorbitol (MACS). A latex agglutination test would then be performed for rapid detection of the O antigen serotype 157. If positive, then all isolates are then sent to the State Health Department for confirmation. EHEC www.studyblue.com EHEC + KLEBSIELLA • • • • • • • • • • usually a lactose fermenter (A/Ag), IMViC= - - + + gram negative rods, non-motile, encapsulated causes enteritis in infants, pneumonia, septicemia, meningitis, wound infections, peritonitis, UTI’s large mucoid colonies - string out - often have yellow centers on MAC plate usually ferments most sugars SPECIES: 1. K. pneumoniae: “Freidlander’s Bacillus” - significant cause of hospital acquired pneumonia 2. K. oxytoca: indole positive 3. K. ozaenae: can be recovered from upper respiratory infections. May contribute to a condition called “ozena” characterized by a chronic, mucopurulent nasal discharge 4. K. rhinoscleromatis: causes a rare form of chronic, granulomatous infection of the nasal passages. Usually characterized by nasal obstruction caused by tumor-like growth. Causes scleroma of nose and pharynx. Can be endemic in Central America, South America and Africa. ENTEROBACTER • • • - gram negative rods, found in soil, water, dairy products and intestines of humans and animals - can cause UTI’s, sepsis, wound infections, nosocomial acquired pneumoniae - differentiated from Kleb. species based on motility (+) and ornithine decarboxylation (+) SPECIES: • • • • 1. E. cloacae 2 most commonly recovered and clinically significant 2. E. aerogenes 3. E. agglomerans - been moved to new Genus: Pantoea meaning “of all sorts and sources” describing bacteria from diverse geographic and ecologic sources; associated with an outbreak of sepsis associated with contaminated IV fluids 4. E. gergoviae - similar to E. aerogenes; most infections are nosocomial (UTI); strong urease + 5. E. sakazakii – (recently renamed Cronobacter sakazakii) is actually biochemically like E. cloacae except with a yellow pigment; associated with neonatal meningitis and sepsis coming from powdered infant formula; best differentiated from E.cloacae by sorbitol fermentation, urea and malonate (E. sakazakii is negative for all) 6. E. taylorae - colonies on MacConkey will develop purple centers after extended incubation • Enterbacter species versus Klebsiella species: ASM MicrobeLibrary.com Bacteriainphotos.com Enterobacter species Klebsiella species HAFNIA • Hafnia alvei - used to be Enterobacter alvei and Enterobacter hafnia; combined 2 species - isolated from various sources; an opportunistic pathogen; smells like human feces CITROBACTER • • • • • • • motile, gram negative rod, usually lactose fermenter often confused with Salmonella and Arizona species because of its close biochemical resemblance key characteristics that differentiate C. freundii from Salmonella are KCN (+), LIA (-), & ONPG (+) *** can be associated with neonatal meningitis; invades through the infant intestinal epithelium and causes systemic disease SPECIES: 1. C. freundii - H2S producer 2. C. diversus/koseri - indole positive 3. C. amalonaticus - malonate negative EDWARDSIELLA • • • • • • • • • IMViC ++ - - Edwardsiella tarda; species of clinical significance; resemble salmonellae - associated with gastroenteritis, wound infections Edwardsiella - glucose fermenter E.coli tarda - motile - indole positive - citrate negative - VP negative - H2S producer www.Bacteriainphotos.com Citrobacter species • PROTEUS, PROVIDENCIA AND MORGANELLA • mostly recovered from UTI infections; common in stool cultures • strains that are able to hydrolyze urea have been associated with the development of renal calculi (kidney stones) in infected patients (will learn biochemical reactions in Unit IV) • Proteus species: demonstrates motility on BAP by “swarming”; species differentiated by indole and ornithine production; both species produce urease very rapidly SUSCEPTIBILITY PATTERNS: • Sometimes the susceptibility patterns of an organism may assist the medical laboratory scientist with the identification of an organism. Below are common patterns seen by the organisms listed: • • • • • • • • • • • • • • • • E. coli S ----> All or most antibiotics Klebsiella R ----> Ampicillin & Carbenicillin Enterobacter R ----> Ampicillin, Cephalothin, Cefoxitin (and other cephalosporins) Citrobacter R ----> Ampicillin, Carbenicillin, Cephalothin Staph aureus R ----> Ampicillin & Penicillin Enterococcus S ----> Ampicillin, Vancomycin (Chloramphenicol & Erythromycin) Beta strep R ----> Amikacin, Tobramycin, Gentamicin (aminoglycosides) & Tetracycline Pseudomonas S ----> Carbenicillin, Tobramycin, Gentamicin, Amikacin species WHEN READING URINE CULTURES: 1. Urine cultures may be finalized after 18-24 hours incubation if one of the following occurs: • - No growth - <= 10 colonies – reported as “No Uropathogens Recovered - > 100,000 Mixed Bacterial Flora (MBF) 2. REHAB PATIENTS (H02 location): REMEMBER: IF A QUAD OR PARAPLEGIC THEN COMPLETE WORKUP WHEN THERE ARE 2 OR MORE PATHOGENS. 3. ** Reminder: To detect Staph saprophyticus: perform a Kirby-Bauer. Set up a 0.5 McFarland Standard of your CoNS. Streak to a small Mueller-Hinton agar and drop the antibiotic “Novobiocin”. Incubate. • * If resistant to Novobiocin, then you have Staph saprophyticus. • If susceptible to Novobiocin, then report “Coag Negative Staph, Probable Contaminant” * Zone size of Novobiocin by KB is: <= 16 mm is Staph saprophyticus (Resistant) >=16 mm is Coag Negative Staph (Sensitive) 4. Urea Positive strains of Coag-Negative Staph: (implicated in production of kidney stones) From pure cultures of CoNS on all men AND women > 18 years old, streak a Urea slant and incubate in non-CO2 for 18-24 hours. (Remember: CO2 alters pH thus altering indicator). True positives are bright pink on 2/3 of slant after overnight incubation or before. Terminology You May Hear In The Lab About Certain Organisms: ESBL’s: • • • Most Escherichia coli , Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Proteus vulgaris organisms are susceptible to extended spectrum cephalosporins, penicillins, and aztreonam. However, on occasion these organisms have spontaneous mutations to occur that produce novel -lactamases that inactivate these extended antibiotics in-vivo and become known as Extended Spectrum Beta-lactamases (ESBL). Strategies for laboratory detection of ESBL-producing E. coli, Klebsiella species and Proteus species include using specific “indicator drugs” (e.g., cefpodoxime, ceftazidime, cefotaxime, ceftriaxone and aztreonam) that will likely detect the presence of an ESBL producer. The MIcroScan System has wells built into their panels that detect these ESBL’s. When an ESBL organism is identified, it should be classified as RESISTANT to all the cephalosporins, penicillins and aztreonam. The carbapenems (imipenem, meropenem, and ertapenem) are active against treating ESBL producing strains. KPC’s/CRE’s: • Klebsiella pneumoniae carbapenemases are serious organisms causing deadly infections that have emerged in health care settings that possess an enzyme that breaks down the action of the “carbapenem” class of antibiotics and show resistance to one or more of the carbapenems. More members of the family Enterobacteriaceae are emerging and thus Carbapenem Resistant Enterobacteriaceae (CRE’s) were born. Usually a combination of antimicrobials (i.e., Colistin and Polymixin B) is used for treatment with a mixed variety of results. These are usually deemed “MDRO’s”- multiply drug resistant organisms. Alphabet Terminology • CAUTI’s: Catheter Associated Urinary Tract Infections • HAI’s: Hospital Acquired Infections a.k.a. “nosocomial” • CLABSI’s: Central Line Associated Blood Stream Infections • • VAP: Ventilator Associated Pneumonia • SIR’s: Standard Infection Ratios References Most of the lecture notes produced in this power point presentation were acquired from: • Required Textbook: Mahon, Connie R., Lehman, Donald C., Manuselis, George,(2015). Textbook of diagnostic microbiology (5th edition). Saint Louis, Missouri: Saunders-Elsevier • Journals/Publications: Notes have also been ascertained using the following: a) Laboratory Medicine b) Advance for Medical Laboratory Professionals • Laboratory Procedures: The procedures used in the following notes with and without pictures were obtained with permission from Carolinas Medical Center-Main, Microbiology Laboratory Procedure Manuals, to be used for educational purposes only by the faculty of Carolinas College of Health Sciences, School of Clinical Laboratory Sciences, Medical Laboratory Science Program. • Hand-outs and Lecture Notes from various members and lecturers of the South Eastern Association for Clinical Microbiologists (SEACM) Annual Meeting Conference Any Questions?