Practical Clinical Pathology PDF

PRACTICAL CLINICAL PATHOLOGY  Sample collection, preservation, & Physical examination  Indication of Urine analysis: 1- Urolithisis 2- Painful Urination. 3- Renal colic. 4- Oliguria or polyuria. 5- Red urine Pyuria. 6- D...

PRACTICAL CLINICAL PATHOLOGY  Sample collection, preservation, & Physical examination  Indication of Urine analysis: 1- Urolithisis 2- Painful Urination. 3- Renal colic. 4- Oliguria or polyuria. 5- Red urine Pyuria. 6- Dehydration or Renal Failure.  Collection of Urine Sample: 1- Voided Urine (Normal Urination) 2- Catheterization 3- Cystocyntesis. 4- Collection Cage 5-External Pressure on U.B External Pressure on Item Voided Urine Catheterization Cystocyntesis Collection Cage U.B spp Small & Large Animal. Small animal Only Small & Lab animal Clean Prepuce &Vulva Suitible for Microbiology Discard 1st drop of urine. Exm. (To avoid M.O in urethra) No urolithiasis ( to Cleaning Periuretheral U.B Must be palpated Urine Stream: - avoid Rupture of UB) area Cleaning Skin 1st → Reflect urethral In small animal by Local Analgesia to Avoid U.B atony in Case of long Suitable for examination of Precautions condition external pressure by decrease Pain. urine distension urine Volume every 24h Midstream→microbiology Thumb & index Sterile Lubricant Contraindication in Examination Large animal by Suitable size of catheter thrombocytopathy patients Last Steam→Reflect U.B rectal Palpation. Avoid rough Handling infection or Prostatic Neglect RBCs in urine Diseases sample up to 5 RBCs/Field Collection Time of Urine Sample is Early Morning. Methods of urine preservation Refrigeration Toluene Thymol crystals Formalin 40% Boric acid Metaphosphoric acid Warmed before Moist suitable Few Crystals 10-15ml One Drop/ 30ml Examination Method urine urine Physical Examination of urine 1- Color 2- Volume 3- Transparency 4- Foam 5- Odor 6- Specific Gravity Foam Transparency or Aspect Normal urine Abnormal Normal urine Abnormal Abundant and slowly Normal Transparent except in Turbid or Cloudy due to presence disappeare due to Proteinuria Horse ( Cloudy due to Ca of Cast, crystals,pus cells …etc Only if Shacked Yellow to green foam due to Carbonate crystals & mucus) bilirubinuria Page |1 Red to Brown Hb uria  Volume Benzedene Test Sample should be collected over 24hrs +Ve -ve Bovine : 20-40ml/kg B.W/24hrs Blood Chemicals Factors affecting volume (Age,climate,exercise, water & feed intake) Anuria: Complete absence of urine ppt uder Micr intact RBCs No ptt (Homogenous) Dysuria: Difficult / painful urination Hematuria Hb or Myoglobin Pollakuria: Frequent Voiding of small amount of urine No ppt Polyuria Oliguria Red ppt Hburia Myoglobinuria High water intake Low water Intake Specific Gravity Parental injection Exercise Chronic interstitial Nephritis Acute interstitial Nephritis Definition Measuring the relative amount of solutes in urine (Failure of kidney to Fever for Long time It indicates ability of Kidney to concentrate urine (Tubular concentrate Urine) Dehydration from GIT disorder Reabsorption) DM & DI (def.ADH) Shock (Hypovolemic fo circ Normal Sp.Gr. is Inversely proportional to urine volume Diuretics Blood ) Pyometra (due to exotoxins) Methods of measuring Sp.Gr Urinometer Refractometer Urine strip Color 100ml of Urine at 20 C Pale Yellow → Polyuria Pink → Congenital Dark Yellow → oliguria Porphyria Normal Sp.Gr 1.020 Yellowish brown → bilirubinuria Red→ Phenothiazine , High Temp 3 C → ↑0.001 Blue → Methylene blue or diazan Phenolphtalein One Low Temp 3 C → ↓ 0.001 Violet → BSP , PSP Reddish brown→ Hematuria Green → Acriflavin treatment Brown → Hburia Brown black → Myogloblnuria (Azuturia) ↑ sp.Gr ↓ Sp.Gt Polyuria except :Diabetes Odor Mellitus Oliguria Normal urine Abnormal urine Renal Failure Cystitis (as a product of Infl ACTH or Corticosteriod Drugs Urinefrous urine due to volatile Strong ammonical odor due to reaction) Isosthenuria Fixed S.G of urine acid action of bacteria on urea Diabates Mellitus (1.008 -1.012) which is equal P a g eMale | 2 Caprine has strong Sweet fruity odor due to S.G of Glomerular Filtrate urinefrous odor of urine ketone bodies ( DM,Pregnancy)  Chemical & Macroscopical examination of urine 1- pH 2- Protein 3- Glucose 4- Ketones 5- Bilirubin 6- Urobilinogen 7- Blood/pH/Myoglobin 8- Ascorbic acid 9-Nitrite 10- Leukocyte esterase 11-Indican 1- pH of Urine ✓ It varies acc. To spp., diet & metabolism. ✓ Normal values: ▪ Horse, Cow, Sheep &Goat ----- 7-9 (Alkaline) ▪ Dog & Cat -------- 6-7 (Acidic) ▪ Pig ------------- 5-9 ▪ Man ------------ 4.8-7 (Acidic) Interpretation of pH of urine Acidic Urine Alkaline Urine 1-Normal in carnivores. 1-Normal in herbivores. 2-High protein diet. 2-High vegetable diet. 3-Starvation (Metabolic acidosis). 3-Cystitis or urine retention. 4-Prolonged muscular activity. 4-Alkaline therapy. 5- Adminstration of some acid salts. 5-Alkalosis (Respiratory or Metabolic). 6- Acidosis (Respiratory or Metabolic). 7-Fever 2- Protein Methods (Strip – Heat and Acetic acid test – Hellers nitric acid – Sulfosalicylic acid test) Heat and Acetic acid test Principle: Proteins are denatured & coagulated on heating to give white cloud precipitate. Method 1. Take 2/3 of test tube with urine, heat only the upper part keeping lower part as control. (Presence of phosphates, carbonates, exudates, proteins give a white cloud formation) 2. Add acetic acid 1-2 drops - If cloud persists then there is protein in urine. - If cloud disappear then there is carbonates/phosphates and exudates. Interpretation of protein of urine ✓ Normally urine is free form protein. ✓ Physiological proteinuria: (Temporary increase glomerular permeability) 1- Excess protein. 2- Neonates. 3- Estrus time in cow. 4- Pregnancy. 5- Emotional stress. 6- Some types of anesthesia. 7- Convulsions. 8- Horse ----- Mucoprotein in urine. Page |3  ✓ Pathological Proteinuria: Prerenal Renal Post renal CNS disturbance Nephritis Urine contaminated with protein: Circulatory Disturbance Nephrosis Pyelitis – Ureteritis – Urethritis Abscess neoplasm Prostatitis – Cystitis Balanoposthitis – Vaginitis 3- Glucose Methods (Strip method – Benedict’s test) In strip method: Glucose + O2 Gluconic acid + H2O2 (In presence of Glucose oxidase) H2O2 +Chromogen Oxidized chromogen +H2O (In presence of Peroxidase) False results of glucose with strip method may be false positive or false negative False negative may be due to: 1) Ascorbic acid. 2) Contamination with formaldehyde or fluoride. 3) Refrigeration of sample. 4) Hyper bilirubinuria. 5) High specific gravity. 6) Protection against humidity. False positive may be due to: Strong oxidizing cleaning agent in urine container. Interpretation of glucose of urine ✓ Normally glucose is absent from urine. (Renal threshold of glucose is 180mg/dl). Glucosuria Associated with hyperglycemia Due to renal tubular damage or dysfunction When blood glucose exceeds 180 mg/dl. It impairs glucose reabsorption, resulting in glucosuria The tubules can’t reabsorb all glucose. without hyperglycemia. (Fanconi Syndrome) ✓ Glucosuria may be transient or permanent Transient Permanent 1- Excessive carbohydrate intake. 1- Diabetes mellitus. 2- Emotional stress. 2- Acute necrotic pancreatitis. 3- General anesthesia. 3- Hyperthyroidism. 4- Systemic stress in ruminant (Cortisol secretion) 4- Hyperadrenocorticism. 5- Glucose infusion, Corticosteroid. 5- Hyperpituitarism. 6- Renal glucosuria (Fanconi Syndrome). 4- Ketone bodies (Acetone/Acetoacetic acid / Beta hydroxybutyric acid) Method (Strip method – Rothera’s) Causes of ketonuria: 1- High fat diet. 2- Ketosis. 3- Pregnancy toxemia. 4- Severe stage of Diabetes mellitus. 5- Prolonged mild fever (off food). 6- Starvation or fasting. Page |4  5- Bilirubin Method (Strip method – Foam test – M.B test) ✓ Normally conjugated bilirubin is absent in urine, but dog, cat urine may contain small amount due to low renal threshold. ✓ Causes of bilirubinuria: 1) Biliary obstruction (Complete or partial). 2) Liver disease. 3) Hemolysis. 4) Intestinal obstruction. ✓ Detection of bilirubin by Vanden Berg Test Serum + Diazo reagent (Aqueous solution) Direct Bilirubin. Serum + Alcohol Total Bilirubin. Total Bilirubin – Direct Bilirubin Indirect Bilirubin. 6- Urobilinogen Method (Strip method – Ehrlich’s reagent). Interpretation of Urobilinogen - Increase Urobilinogen (1) Obstructive jaundice (Intrahepatic obstruction). (2) Hemolytic Jaundice. - Absence of Urobilinogen (1) Biliary obstruction. (2) Enteric antibiotics. (3) Impaired intestinal absorption (Severe Diarrhea).\ (4) Polyuria. 7- Blood/Hb/Myoglobin Interpretation ✓ Hematuria (Intact RBCs) 1) All inflammation in UGT Uro-Genital Tract (Intact RBCs in urine sed. E clear urine). 2) Urolithiasis. 3) Trauma. 4) Estrus in bitch. 5) Parasites. ✓ Hemoglobinuria (Free Hb in urine) 1) Post-parturient Hburia. 2) Bacillary Hburia. 3) Cl.perfringens type A. 4) Leptospirosis. 5) Blood parasites. 6) Hemolytic diseases. 7) Incompatible blood transfusion. 8) Chemical agents as toxic plants. ✓ Myoglobinuria As in case of: 1) Azoturia in equine. 2) Severe rhabdomyolysis. Page |5  Benzedene test +Ve result -Ve result Blood Chemical Then do centrifugation No ppt. (Homogenous) PPt. under microscope intact RBCs (Hb or myoglobin) --- (Hematuria) Add 5ml urine + 2.5ml ammonium sulphate Red ppt (Hburia) No ppt (Myoglonuria) 8- Ascorbic Acid 9- Nitrite ✓ Method (Test strip) ✓ Interpretation Indicate presence of bacterial infection so reduce nitrate into nitrite. Any pink color is considered +Ve 10- Leukocyte esterase ✓ Positive results indicate presence of pus cells (pyuria) 11- Indican ✓ Its derivative of indol metabolism caused by protein putrefaction in intestine. ✓ Interpretation (1) Malabsorption. (2) Constipation. (3) Enteritis. Page |6  Macroscopical Examination of urine (Cast&crystal) Macroscopical Examination of urine Has Great Clinical Importance It should made shortly after Collection, Where Delayed Exam can Cause 1- Degeneration of Cells. 2- Bacterial Proliferation. Normal Urine Contains Small Amount of Mucous Threads, Epithelial Cells ,WBCs , RBCs. Normal urine of Horse Contain Ca-Carbonate Crystals and Mucous Threads. External Objects as Parasitic Eggs, Fungal Spores & Other Organics Due to Fecal Contamination. Classification of Urine Sediments (Organized , Unorganized) Organized urine sediments Leukocytes Erythrocytes Epithelial Cells Normally Few RBCs present (0- 3/HPF). If Number of RBCs >10 RBCs/HPF→ Normally few WBCs present (0- Hematuria. 5/HPF). Interpretation:- If no. of WBCs (Pus cells) 1. Inflammation or necrosis of urogenital Increased→ pyuria. tract. >10pus cells/HPF inflammation 2. Damage of Capillaries. or urogenital tract or kidney. 3. Renal Infarction./ Urolithiasis.  Types of epithelial cells acc 4. Hemostatic disorder. Specimen by cath. Or cysto. So histological structure. 5. Violent exercise. WBCs from UB or ureter or 1- Large Squamous epi→ 6. Trauma or Rough Cath. Pelvis or Kidney. 7. Sample collected during bitch estrous. Largest cell lining urethra Decrease associated with Pyuria ❖ Appearance→ Round or oval Internal or Vagina. are Nephritis , Pyelonephritis , structure. urethritis and cystitis. 2- Transitional epi→ lining Morphology of RBCs differ acc to Under MS → bigger than RBCs Sp.Gr. of Urine: - renal pelvis, ureters, U.B. & Granulated. 1) High SP.Gr→ Shrinked/Crenated 3- Renal epi→ small round If they are not well preserved 2) Normal→ (1.020-1.035) Biconcave. appearance of pus cells cells lining renal Tubules. 3) Low→ Ballooned/ Spherical or Lysed changes→ degeneration of (Ghost Cell) Nucleus & ppt of crystals on We Can Detect Location of cell surface. Hemorrhage acc to presence of In Alkaline Urine Cells are Blood in urine stream: - swollen & Very Granular. ✓ 1st stream→ Urethra,Vagina,Penis, Prostate. ✓ Uniform all over steam→ Kidney , Ureter ✓ In Last Stream→ U.B. Page |7  Casts Bacteria  Presence of Cast in The Urine Indicates pathological condition in the Kidney.  Casts are formed only in the loop of hannel or DCT or Collecting duct (Tubules lining are sloughed & Normal urine is free from bacteria in U.B so appear in urine). sample is collected aseptically by Cystocentesis or  Casts to be formed Cath. 1. Proteinuria No Significance to presence of bacteria in urine 2. Common in Concentrated acidic urine (For sample standing at room Temp. protein denaturation and ppt) less in Alkaline. Presence of yeast or fungus due to mycotic 3. Stagnation or stasis of urine in tubules. infection  N.B: Casts are not formed in urine of cattle. Presence of protozoa and parasitic ova due to stool  Significance of casts: - Contamination 1- Localized sign of Renal tubular disease. Sperm. 2- NO. of Casts not indicate the severity of disease as in renal failure only few NO. of casts are found. Cast appearance Interpretation Retractile Slight Renal disease Hyaline Cast Translucent Reversible smooth Moderate Renal disease Granular Cast Reversible Waxy Cast Sever Chronic renal failure irreversible Normal in Dog & Cat (Lipuria) Fatty Cast Man (D.M Fatty Change in Renal Tubules obesity) Erythrocytic Cast Sever Hematuria Leukocytic Cast Sever Supportive Inflammation Epithelial Cast Mentioned Before Automated urine analysis ✓ No Centrifugation ✓ Sp.Gr Measured by Mass gravity meter ✓ Chemically, Spectrophotometer ✓ Microscopically, Automated intelligent microscope with camera attached Page |8  Unorganized Sediments Source: exo, endo Significance: urolithiasis  Leucine, Cystine & tyrosine crystals→ severe liver disease.  Lipuria.  Bilirubin crystals.  Ca- Carbonate crystals & Mucous. Acidic Crystals Type Appearance Interpretation  Rosette , Rhombic form Increased in Cellular turnover yellowish brown Dissolved by (Lymphoma, Leukemia, Gout) Uric Acid NaoH So Increase in Porteinuria metabolism and then increase in Uric Acid  Na, Mg, K, Ca salts  Granular Sand Amorphous Urates  Yellowish Brown  Dissolved by Heat  Envelope Shape Ca- Oxalate  Vary from Large to small  Dissolved by HCL  Plate, Needle, Rhombic, Cubes, Granular.  Yellow Brown to Reddish brown Bilirubin Toxic or Obstructive J.  Dissolved by Acid or Alkali acetone or chloroform  Insoluble in alcohol & ether  Plate form or needle conc at Hippuric center  Fine delicate needle in clusters (colorless to yellow) Tyrosine  Disssolved by actic acid or alkali Liver Disease or heat  Insoluble in water , acetone.  Car Wheel (yellowish brown) Associated with Tyrosine , so appear Leucine  Dense and highly retractile in severe Liver disease  Dissolved by NaoH. Genetic disorder Pyelonephritis  Hexagonal plate Cystine Heavy Metal  Dissolved by HCL or ammonia Nephrotoxicity Renal tubules acidosis Page |9  Alkaline Crystals Type Appearance Interpretation ✓ Granular sand Amorphous phosphate ✓ Dissolved by acetic acid ✓ Coffin Lid Shape (Prism Like) Triple Phosphate ✓ Colorless Chronic urinary tract Infection Mg, Ammonia, Phosphate) ✓ Dissolved by 10% acetic. ✓ Spiny Apple (Thorn apply) ✓ Yellow Brown Ammonium Blurate ✓ Dissoloved by acetic acid , heat at 60 & NaOH on standing ppt ✓ Large colorless or Yellowish spheroids with radial striations Ca-Carbonate ✓ Or small Dumbbel shape ✓ Dissolved by Acetic acid. P a g e | 10  Urolithiasis  Definition: Formation of Urinary Stone (Calculi).  Source: 1- Food 2- Water 3- Metabolic  Factors affecting calculi formation: 1- Vit.A deficiency (epithelial desquamation). 2- Hapercalciuria in parathyroid disease. 3- Hyperuricemia (in gout disease). 4- Hypercrystalloiduria. 5- Cystitis and urine retention (infection). 6- Acidic urine develop acidic calculi as xanthine & oxalate. 7- Presence of focci of calcification in kidney. 8- Hyperoxalouria as in excessive oxalate ingestion, iliac disease & steatorrhea. Steatorrhea ✓ Stool: Clay Color, Bulky, Offensive odor. ✓ Occur in: 1. Obstructive Jaundice 2. Chronic Pancreatitis Types of Urolithiasis Name Primary Calculi Secondary Calculi Cause ✓ Hypercrystalloid content of urine→ ppt of ✓ Presence of solid mass act as nidus for crystals stone formation as necrotic tissue or ✓ Stone are mixed as urates, uric acid..etc inflammation, so crystals precipitated around the nidus Common Primary Calculi item Urate stone Ca salts stones Composition Uric acid + ammonia & Urates Ca salts (oxalate & phosphate) ✓ Round, Translucent, Brown, Smooth ✓ Hard, Vary in color yellow to dark brown & Hard. Characters ✓ Rough Surface with sharp spikes so it is ✓ This Calculi make pressure on kidney very painful. causing hydronephrosis. Rare Primary Calculi item Cystine Stones Xanthine stones ✓ Yellowish brown ✓ Reddish brown or yellowish Characters ✓ Very Firm ✓ Oval, Smooth ✓ Presence in Live Disease ✓ Friable Secondary Calculi Composition Ca phosphate + Mg & ammonium phosphate + Variable amount of Ca Carbonate Characters ✓ White ✓ smooth ✓ soft ✓ Friable P a g e | 11  Fecal examination ✓ Aim: Mainly for detection of parasites. (1) Collection of sample ✓ Precautions: 1. Sample must be labeled. 2. Sample must be fresh. 3. Sample must be of proper quantity (5gm from small animals – 10gm from large animals). 4. Container must be clean and clear. ✓ Methods: 1. Direct from the ground (suitable for all 3. Rectal enema in small animals. animals). 4. Sterile fecal spoon for microbiological 2. Rectal palpation in large animals. sample. (2) Preservation 1. Eggs and oocysts are preserved in 40% formalin (1 part of formalin : 4 parts of feces). 2. Trophozoites mustn’t be preserved because diagnosis of them depending on the motility (examination without any delay). (3) Examination a) Macroscopic examination − Normal odor is fecal odor due to presence of scatol. 1. Odor − Fetid odor indicates GIT infection. 2. Consistency Solid → Constipation Semisolid → Normal Watery → Diarrhea − Normal color is brown due to presence of stercobilin. − Dark brown indicates hemolytic jaundice or iron therapy. 3. Color − Clay color indicates steatorrhea (gray color with offensive odor due to putrefaction of proteins). − Normal fecal matter doesn’t contain blood. 4. Blood − Bright red blood → Rectal hemorrhage. − Brown to black blood → Gastric or intestinal hemorrhage. − Normal fecal matter doesn’t contain mucus. 5. Mucus − Its presence indicates catarrhal enteritis. Gross parasites can be found in fecal matter as ascaris, strongylus, segmented 6. Parasites tapeworms. P a g e | 12  b) Microscopic examination 1. Qualitative tests (indicate presence or absence of the parasite) Item Sedimentation test Floatation test High specific gravity diluent will make Low specific gravity diluent will make eggs Principle eggs float on the surface easing their sink on the bottom easing their collection. collection. Low specific gravity as: High specific gravity (1.200-1.250) as: 1. Tap water or saline. 3. Saturated salt solution (NaCl). 2. Acid either. 4. Concentrated sugar (Sheather’s Diluent solution). 5. Zinc sulphate. 6. Sodium nitrate. a) By water or saline: 1. Add 1-2gm feces to 15-30ml of diluent. 1. Add 1-2gm feces to 15-30ml of diluent. 2. Centrifuge. 2. Sieve by gauze or tea sieve. 3. Remove supernatant, collect part of 3. Fill the tube to the end and cover by sediment, and examine under cover slide and wait 60min till noting microscope. convex surface, eggs float and touch the Steps b) By 5% acetic acid or HCl either: cover slide. 1. Add 1-2gm of feces to 15-30ml of 4. Or fill the tube and centrifuge acetic acid, sieve, then add either of (1000rpm/5min) till floatation of eggs, equal amount to mixture. collect them by bacterial loop and 2. Centrifuge at 2500rpm/1-2min. examine under microscope. 3. Remove supernatant, collect part of sediment, and examine under microscope. Used for Some nematode eggs and trematode eggs Oocytes, cestodes, eggs of nematodes Not representative, so –ve results Disadvantage --- frequently occur. Item Direct smear Baermann method High specific gravity diluent will make Principle --- eggs float on the surface easing their collection. Saline (for trophozoite to keep it motile and Diluent Warm tap water or saline intact) or tap water. Steps After movement of larvae from feces to warm water, untie the clamp, and put water in Petri dish and direct examination under microscope or put one drop and examine it. Used for Specific for nematode larvae Not representative, so –ve results Disadvantage --- frequently occur. P a g e | 13  2. Quantitative tests (give accurate counting of parasites) 2. a) McMaster:  Steps: 1. Add 2gm of feces to 28ml of water. 2. Aspirate 1ml of mixture by Pasteur pipette the add 1ml of Sheather’s solution. 3. Shaking well. 4. Filling 2 chambers of McMaster slide (each slide filled with 0.15ml). 5. Wait 20min then count.  Calculation: 𝐸𝑃𝐺 (𝑒𝑔𝑔 𝑝𝑒𝑟 𝑔𝑚) = 𝑁 × 100 × 2 (𝐼𝑓 𝑌𝑜𝑢 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑜𝑛𝑒 𝑐ℎ𝑎𝑚𝑏𝑒𝑟). 𝐸𝑃𝐺 (𝑒𝑔𝑔 𝑝𝑒𝑟 𝑔𝑚) = 𝑁 × 100 (𝐼𝑓 𝑌𝑜𝑢 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑡𝑤𝑜 𝑐ℎ𝑎𝑚𝑏𝑒𝑟). 2. b) Stool egg count:  Steps: 1. Add 4gm of feces to 56ml of NaOH (N/10). 2. Shaking well. 3. Add 0.15ml by Pasteur pipette of micropipette on slide and count.  Calculation: 𝐸𝑃𝐺 (𝑒𝑔𝑔 𝑝𝑒𝑟 𝑔𝑚) = 𝑁 × 100 P a g e | 14  Clinical microbiology ❖ Routine diagnostic microbiology in Veterinary medicine is divided into: 1- Isolation and identification of infectious agents. 2- Culture and sensitivity test for selection of the proper anti-microbial agents in Treatment. 3- Serological tests for diagnosis of most common infectious diseases. ❖ Sample collection for microbiological exam: 1- Technical efficiency of the individual performing the test ⮑ Know how & when to take a sample. ⮑ Select the confirmatory test for diagnosis. ⮑ Interpret the results. 2- Samples must be representive for the disease. 3- Collection of sufficient amount of the sample. 4- Avoid Contamination (To avoid Misdiagnosis). 5- Proper handling of the sample. 6- Samples must be collected before administration of antimicrobial agent. Common samples collected in diagnostic microbiology item Blood Urine Feces Wound, Abscess, throat, ear, Eye swab ✓ Shaving of area ✓ Collect by ✓ Sample should Closed abscess Opened abscess over the vein. cystocentesis or be directly 1. Clear around. Collected using ✓ Wash with soap Catheterization. obtained from 2. Collecting the sterile cotton swab & Water For Man: rectum by: pus by sterile & should be taken ✓ Let skin dry 1- Washing with 1- Rectal syringe under from deeper part to ✓ Draw 5-10ml soap and water. Palpation. complete aseptic exclude existing blood with 2- Exclude 1st 2- Fecal swab. condition. Precautions M.Os sterile syringe stream. ✓ Then put in 50- 3- Sample from ✓ Refrigeration of Throat, ear & eye swab the sample if 100ml of rich middle stream. ⮑ Swabs should be collected carefully nutrient broth ✓ Collected in not examined for culture. within 1hr. media. sterile tube. ⮑ After swabbing , swab should be ✓ Refrigeration of placed in test tube containing 1-2ml the sample if not sterile saline or broth to avoid dryness examined within of swab as M.O won't survive in the 1hr. absence of moisture. ❖ NB: For Puncture fluids synovial, pleura, peritoneum): Shaving the area + Washing + sterilization by alcohol + collect 5-10ml fluid on 50-100ml rich media by sterile syringe. Types of culture media Liquid media Solid media selenite F Nutrient broth Simple enriched selective differential broth tetrethionate peptone water broth Meat infustion loffeler's broth serum P a g e | 15  item Solid media Liquid media - For isolation & identification of pure culture Provided as a base of nutrient ❖ Simple Media: medium it supports the growth Used in routine bacterial exam of swabs, fluid, tissue. of MO after original isolation on 1- Nutrient agar medium: solid media. For maintenance of M.Os. Rarely used for original isolation. ❖ Nutrient broth Base of other media as enriched or selective media. - Simple media for growth of 2- Nutrient gelatin medium: most pathogenic MOs To identify proteolutic M.O as Vibro cholera. ❖ Enriched media: ❖ Selenite F-broth Complex media & essential for growth of some MO - Salmonella & Shigella I. Blood agar medium. 1) Prepared by adding 5-10% mammals sterile blood to a nutrient ❖ Tetrathionate broth agar at 45C. 2) For isolation of most aerobic M.Os. - Salmonella typhi & S.paratyphi 3) Used for differentiation of some MO according to their action on blood: ❖ Leffeler serum ⮑ Complete hemolytic (B hemolytic) as in staph aureus , - Corynebacterium diphtheria strept.Pyogens , strep equi. ⮑ Partial hemolysis (a-hemolysis) as in Strept Vridans. ❖ Pepton water broth ⮑ No Hemolytic (Gamma hemolysis) as in staph epidermidis. II- Chocolate agar medium. ❖ Meat infusion broth Heated blood agar. For isolation of hemophilus. ❖ Selective media. Are media with chemicals or dyes or antibiotics which allow the growth of certain bacteria & inhibit growth of other. ‫مهم جدا جدا‬ 1. Lowenstein Jensen medium for Mycobacteria 2. Blood tellurite medium for Corynaebacterium diphtheria. 3. T.C.B.S medium for Vibrio cholera. 4. Thayer martin medium (modified chocolate agar) N. Gonorrhoeae. 5. Desoxycolate citrate agar (DCA) for Salmonella paratyphi. ❖ Differential media. a. MacConkey agar medium Has neutral red indicator to differentiate enterobacteriacae into: Lactose fermenter which give pink colonies & non Lactose fermenter which give Pale yellow colonies b. Triple sugar iron agar (TSI) To differentiate between salmonella , E.coli, & pseudomonas aeruginosa. Methods of bacterial Cultures P a g e | 16  Preparation of smear of microscopic examination 1-Direct Smear from pathological specimen Used as a Part of any microbiological examination. Applied on: - Urine sediment - Exudates - Milk - Cerebrospinal fluid Technique: Roll sterile cotton swab from the sample over the surface of clean glass slide. 2-Smear from Broth culture: 3-Smear from Solid media (from colonies on solid media): Steps ✓ Under complete aseptic ✓ Put 2 drops of water on center of clean glass condition shake the broth tube. slide. ✓ Put 2 drops from culture tube ✓ Add loopful of colony from culture by by sterile loop on clean glass sterile loop. slide and spread. ✓ Mix bacterial colony with water drop and ✓ Allow air dryness spread on the slide. ✓ Fix the smear with heating ✓ Fix the smear by heat. (pass the slides about 3-5 times forth and back through the flame of Bunsen). N.B. Heat: o Killing bacteria. o Adhere it to glass slide. ▪ Microscopic examination of M.O: 1. Wet preparation method. Differential 2. Stained smear method. Simple stain stain Wet preparation method o Significance: For examination of living & motile M.O. o Steps: - Put drop of liquid or broth on Glass slide. - Apply cover slid - Examine at low then high power to detect morphology and motility. P a g e | 17  Stained smear method a. Simple stain b. Differential stain Used for o Detection the presence or I- Gram stain II- Zeihl-Nelsen absence of bacteria. -If present - stain --Detect its morphology, The most common Specific for acid arrangement and size. stain used in fast bacteria as o Examples: - Methylene Blue - Identification of Safranin - Crystal violet. bacteria. Mycobacterium tuberculosis. principle When bacteria retain This bacteria has the basic dye after high fat content so TTT with by using strong decolorizing agent carbolfuchsin and as alcohol, so it is heat →the dye gram positive. penetrate the While the gram- organism. negative bacteria decolorize the stain. Steps - Basic stain: Crystal - Basic stain: Strong violet carbolfuchsin - Decolorizing agent: - Decolorizing agent: alcohol 95% 25% sulfuric a. - Counter stain: Diluted - Counter stain: carbolfuchsin. methylene blue. Mordant: iodine Mordant: gram. application of heat. Gr+: violet or blue. Acid fast: pink. Gr- : pink or red. Non-acid fast: blue. P a g e | 18  Culture &Sensitivity, Serological Tests Methods of Sensitivity test: 1. Disc diffusion. 2. Tube dilution. method Disc diffusion Tube dilution The aim => 1- Isolate infectious agent. 1. Two fold dilutions of the antibiotic are prepared. steps 2- Determine proper therapy. 2. A constant volume broth culture of the bacteria is added The test is done => to each tube. o Directly from the pathogenic samples as (sputum, pus, urine). 3. Two control tubes should be included: Or (From pure culture isolate). 1. Prepare culture plate containing routine culture media ❖ One tube containing the antibiotic with no bacteria. (Nutrient or blood agar). 2. Cover plate agar surface with the sample using either: ❖ One tube containing the bacteria with no antibiotic. -Swab. -If the sample was in broth so flooding the plate surface with the broth culture for 30 sec and remove excess. 4. After incubation at 37 degree for 18-24 hours, all 3. Dry the culture plate for 30 minutes or on bench for 1 tubes are checked for turbidity. hour. 4. Put the selective antibiotic discs to the surface of the plate (The space between every disc must be not less than 2 cm) ✓ MIC (minimal inhibitory concentration): 5. Put the culture plate in the incubator at 37C/24hr. The tube with lowest concentration (highest dilution) 6.Measure the diameter of the zones of inhibition of which inhibit bacterial growth and show no visible bacterial growth. turbidity. 7. The absence of inhibition zone around antibiotic disc indicates resistance of organism to these antibiotics. ✓ MBC (minimal bactericidal concentration): 8. According to the diameter of inhibition zone we report Tube with highest dilution which kill bacterial growth. as: - Sensitive (respond to antibiotic with ordinary dose) or - Moderate (respond to high dose of antibiotic) or - Resistant (No response to this antibiotic) P a g e | 19  Serological Tests laboratory diagnosis of disease in a patient’s serum using specific Ab for a specific Ag or vice versa. Serological tests based on Abs specifically binding to Ag: - Ag of known identity will react with Ab in an unknown serum sample - Known Ab can be used to detect Ag in serum. Types of common serological tests Agglutination tests Neutralization tests Haemagglutination reactions Labels Complement fixation Immunoassays tests(CFT) Direct agglutination Latex agglutination Coomb’s test Indirect haemagglutination ELISA (Antiglobulin test) test Widal C-Reactive protein (typhoid) (CRP) Direct Coomb’s test Direct and indirect Immunoflourescence Brucella agglutination Rheumatoid factor (RF) Indirect Coomb’s test Radioimmunoassays Antinuclear antibodies (ANA) 1- Agglutination tests 1. Direct agglutination: Drop of Ag + one drop of Ab (serum) → agglutination within 2minutes. 2. Latex agglutination: Ab coated on latex beads and when add Ag (serum) agglutination is noticed. a. C-Reactive protein (CRP): Is acute phase protein in serum of patients suffering from acute inflammation as Rheumatic arthritis. b. Rheumatoid factor (RF): For diagnosis of Rheumatoid fever c. Antinuclear antibodies (ANA): For diagnosis of systemic autoimmune diseases as systemic lupus erythromatosis (SLE). 2- Haemagglutination reactions Direct Coomb’s test: Done on infant RBCs in case of erythroblastosis fetalis. +ve test means patient has Abs (anti Rh factor). Indirect Coomb’s test :Used to detect Anti Rh factor in maternal serum. P a g e | 20  b. Indirect haemagglutination test : - For diagnosis of some viruses, Toxoplasma and Bilharziasis stimulate immune system to produce titer of antibody in the serum. -Serial dilution of serum in microtiter plate ---- then add certain amount of Ag coated on washed RBCs to each well. The Ab titer is the 1st well showing complete ppt of RBCs in the bottom. 3- Neutralization Tests - Toxin-antitoxin neutralization - For diagnosis of Antistreptolysin O (ASO) for diagnosis of Rheumatic fever. 4- Labels Immunoassays a. ELISA: - For diagnosis of some viral, bacterial & protozoal diseases through: estimation of specific Ab titer (IgM & IgG) and anticardiolipin antibodies (ACAs) - ACAs associated with venous & arterial thrombosis,thrombocytopenia and recurrent fetal loss. b. Direct and indirect Immunoflourescence: - For diagnosis of Antineutrophil cytoplasmic antibodies (ANCA). (ANCA are group of autoantibodies associated with autoimmune necrotizing diseases, systemic vasculitides & idiopathic glomerulonephritis). c. Radioimmunoassays: Method of choice for hormones estimation. 5- Complement Fixation Tests (CFT) - Used in research work. - Rare to be used in lab. diagnosis. P a g e | 21