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Done by: Sumiya Talal Al harbi Under supervision of specialist: Majed Abdulhamed Alshaer KING ABDULLAH MEDICAL CITY HOLY MAKKAH 30, NOV2016 WELCOM TO MICROBIOLOGY Internship year This book provides a basic information about microbiology section which collected from different resources. 1|Page FOR MORE INFORMATION CONTACT: [email protected] Table of Contents Introduction about microbiology ................................................................. 4 Methods of identification: .....................................................................................................5 (basic bacterial classification) ..................................................................... 17 Microbiology section in KAMC: ................................................................... 19 First: reception and receiving area ..................................................................................... 22 Second: respiratory bench .................................................................................................. 24 Third: blood bench area ...................................................................................................... 28 Forth: urine and stool bench .............................................................................................. 29 Classification and testing of Antibiotics ...................................................... 33 Antimicrobial susceptibility testing (AST) ...................................................35 Minimum inhibitory concentration (MIC) .................................................. 36 E Test Method: ............................................................................... 37 2|Page Objectives: -To know the basic of bacteriology including, taxonomy, bacterial classification, different diseases, morphological and microscopic identification of bacteria. -To understand the type of pathological and normal flora of bacteria. - To be able to perform all the practice in each bench of microbiology. -To understand the mechanism of action of antibiotics against each bacterium. -To know the principle of instruments used in microbiology section. The message: To facilitate the practical and theoretical information and handled to each intern. Acknowledgment: I would like to thank Mr. Majed Al shaer for helping the preparation of this booklet and providing helpful suggestions and encouragement throughout our internship and our life in general. 3|Page Introduction about microbiology Microbiology is: study of living organism which can't be seen by necked eye and seen with the help of microscope. Taxonomy is: The since of biological classification including, nomenclature and identification of organism. Classification is: the arrangement of organisms into groups (e.g. families, genera) on the basis of similarities or relatedness in their various characteristics, which includes: — Morphology — Staining properties — Cultural characteristics — Biochemical reactions — Antigenic structures — Genetic relatedness Morphology of bacteria: Cell Shape: -Bacteria have a wide variety of sizes and shapes. 4|Page - bacteria that possess cell walls exist in three basic morphologic forms: Spherical (round) cells or cocci Rod shaped cells or bacilli Spiral shaped cells or spirilla Cell Size: Bacteria are 0.2 to 2 μm in diameter and 1-6 μm in length.2 Methods of identification: MICRO SCOPIC CULTURE CHARACTER ISTICS 1- Microscopic identification: 5|Page BIOCHEMI CAL REACTION S SEROLOGIC TESTS MOLECULA R METHODS Stained Preparations Unstained Preparation Wet Mount Simple methylen e blue& carbol fuchsin Gram’s Staining Negative Staining ZiehlNeelsen Staining for mycobactiru m tuberclosis Staining Methods Giemsa Staining Albert’s Fluorescent e.g.coryneb acterium sp. Spore Flagella 1-Unstained (wet film) procedure: usually used to identify the fungi from bacteria. 40X power 6|Page 2- Stain procedure (gram staining): *The Gram’s stain divides the bacteria into two groups: -Gram-positive bacteria (Blue or purple colour) -Gram-negative bacteria (Pink or red colour) *The Gram stain requires the use of: 1- Primary stain - Crystal violet 2- Mordant – Iodine 3-Decolourizer - Alcohol and 4- Counter stain - Safranin Procedure: 1 mint 1 mint seconds 1 mint *Function of iodine: fix the crystal violet on the wall of bacteria. Factors affecting the result of gram staining: -Over depolarization -improper fixing the smear -over heating - the freshness of the reagents 7|Page Gram staining result: Positive gram stain seen under 100x oil negative gram stain seen under 100x oil 2- ziehl-neelsen staining: Principle: Mycobacterium species, are coated with a thick lipid material (e.g. mycolic acid) in their cell walls and keep the basic dye (basic fuchsin) despite of acid alcohol treatment and considered as acid fast (AFB). Procedure: 1 mint 8|Page seconds 2 mint Result seen under 100x oil immersion: Acid fast bacilli >> commonly sample used is sputum to see mycobacterium tuberculosis that cause TB. 3- Culture media: An artificial culture medium should contain all the nutrients i.e. -sources of carbon -sources of nitrogen -sources of minerals -sources of essential vitamins and water that are needed by the bacteria for its growth and multiplication in the laboratory. Forms of culture media: -Solid media(agar) -Liquid (broth) -Semi-solid media 9|Page Type of media indicator SAB (Sabouraud Dextrose Agar) _____ Blood agar _____ XLD agar (Xylose lysine desoxycholate) _____ -Type of selective media which select the growth of salmonella and shigella and inhibit the growth of others. - salmonella produce a black colony due to production of H2S - shigella will be colorless on agar -sample used: stool ____ Used as a culture media for streptococcus agalactia in maternal infection. Todd Hewitt broth 10 | P a g e information -Used for fungal culture -contains antibiotics that inhibits growth of other organisms including: chloramphenicol& gentamicin - low PH acidic media -Type of enriched media which specific substance is added - shows type of hemolysis which are: Alpha> partial hemolysis Beta > complete hemolysis Gama> no hemolysis pictures Chocolate agar ____ Type of enriched media that contain lysed RBCs, X and V factors that required for H. influenza growth. Mannitol salt agar Phenol red -Type of selective media for staph. Aureus. - used for MRSA screening test (methicillin resistant staphylococcus aureus). -nowadays, oxacillin used instead of methicillin. -MRSA produce a yellow colony on mannitol agar due to fermentation of mannitol. - Other staph produce a pink color. -contain a high concentration of salt 6.5% which inhibit other bacterial growth. - if staph grow in culture this indicate resistant to oxacillin. - sample used: groin, axilla and nasal swab. MacConkey's agar Neutral red -Type of selective media for gram negative bacilli bacteria and differential media for lactose fermented (pink color) and non-lactose fermented organism (yellow color). -contains: crystal violet and bile salt which inhibit growth 11 | P a g e of gram positive bacteria - proteus can grow but it will not produce swarming due to bile salt. - used for CRE (carbapenem resistant enterobacteria), CRP (carbapenem resistant pseudomonas) and Acinetobacter resistant. Selenite broth ______ Bile esculin agar _____ Thioglacte broth ______ Thiosulphate citrate bile salt sucrose (TCBS) agar 12 | P a g e -Enhancement media for salmonella and shigella growth. -after 24h of incubation a sample taken from selenite and culture on XLD agar this called sub-culture process. -Type of selective media for enterococcus organism which produce a black color on agar. - used for VRE screening test (vancomycin resistant enterococcus) -sample used: rectal swab. -Mechanism of action: the organism will react with esculin hydrolysis Product+ ferric iron black color. Used for anaerobic organism ______ Sample used: body fluid. Bromothymol Type of selective media for blue vibrio's and differential media which differentiates sucrose fermenting (yellow colonies) from non-sucrose fermenting Vibrio's. Molar Hinton agar _____ Thayer martin agar _____ 13 | P a g e -V. cholera produce yellow colony - V. Para hemolytic produce colorless colony. -TCBS contain sodium thiosulfate which inhibit other bacterial growth. - alkaline PH. -Simple media used for manual sensitivity antibiotic test. -ingredient: beef, peptone and starch. Used for H. influenza sp by utilizing X, V and XV factors. - X factor> hematein - V factor >NAD (Nicotinamide adenine dinucleotide) -H. influenza & H. aegyptius utilize both V&X factors -H. parainfluenza utilize only V factor -H. ducreyi utilize X factor -Selective media for Neisseria sp. -contains antibiotics that inhibits other bacterial growth including: Colistin> to kill gram negative bacteria Vancomycin> to kill gram positive bacteria Nystatin> to kill fungi and yeast Sulfa methylene and\ trimethoprim to inhibit proteus growth. Ingredient: molar Hinton, chocolate and 5% of antibiotics. Einstein lactose Bromothymol -Used for urine culture electrolyte defect blue -Selective for Enterobacter (CLED) and E, coli which produce a yellow shadow Chrome agar -For colorful microbial detection. -Used mainly for multi drug resistant organism including: Acinetobacter> produce green color VRE> red color CRE> purple color -Biochemical test Test for enzymes including: 1- Catalase test 2- Coagulase test 3-Oxidase test 4-Urease test Catalase test: Principle: To determine the production of catalase enzyme by the organism, which breaks down H2O2 into H2O and O2. Control Organisms: +Ve control – Staphylococcus aureus -Ve control – Streptococcus species 14 | P a g e Result: Rapid production of (bubbles). Coagulase test: Principle: To test the ability of an organism to clot plasma by coagulase enzyme. Two type of coagulase test: Free coagulase (tube test): which converts fibrinogen to fibrin by activating coagulasereacting factor present in plasma. • not commom used Bound coagulase (slide test): which converts fibrinogen directly to fibrin without requiring coagulase-reacting factor . It is detected by clumping of bacteria in the rapid slide test • commonly used Control Organisms: +Ve control – Staphylococcus aureus -Ve control – Staphylococcus epidermidis Result: Oxidase test: 15 | P a g e positive negative Principle: To detect the presence of oxidase enzyme, which oxidizes cytochrome by molecular oxygen. Interpretation: Positive: deep purple within 10 sec. Negative: No color change. This test used to identify gram negative non-lactose fermented bacteria +VE >> pseudomonas -VE>> Acinetobacter & protues Result: Urease test: Principle: To detect the ability of organism to produce the urease enzyme, which splits urea to ammonia and carbon dioxide. Interpretation: Positive – Pink color >>Klebsiella sp, Proteus spp., Helicobacter pylori Negative - Pale yellow>>Escherichia coli negative positive Result: (basic bacterial classification) 16 | P a g e bacteria classification based on gram stain reaction Gram positive Gram negative bacilli cocci bacilli cocci Neisseria Moraxella H. influenza streptococcus staphylococcus Corynebacterium Catalase test diphtheria (normal flora) on negative strep Enterbacteriac eae skin Positive staph Coagulase test Positive >staph.aures Negative (CNS) Novobiocin test Resistant>s. saprophytic cause UTI infection 17 | P a g e Sensitive> s. epidermis (normal flora) streptoccocus classified according to hemolysis alpha hemolysis greenish color on blood agar due to conversion of Hg to> methemoglobin beta hymolysis gamma hemolysis classified according to lancefield group to: Group D Enterococcus - hemolytic, Bile optichin sensetivity test sensitive> s. pneumonia (mobile on bile solubility test) resistant > s. viridans( normal flora) Staph. aureus Group A> s. pyogenespinpoint on blood agar. wide zone of hemolysis. causes pharyngitis. Bacitracin test> sensitive aesculin hydrolysis Positive. Small gray colony on blood agar, and black on bile aesculin agar. Group B> s. agalactia normal flora in female genital tract. - big colony on blood agar. -narrow zone of hemolysis. Bacitracin test> resistant (CAMP) Identification test for s. agalactia. Test showing the arrow-shaped zone of enhanced hemolysis (positive result) of Streptococcus agalactia (group B) 18 | P a g e The streak plate technique: A streak plate technique is used to obtain single isolated colonies from a clinical specimen (sample). 1 4 2 3 After incubation, single isolated colonies should be visible within the fourth zone. A small portion of sample is taken on a sterile loop and streak on an agar medium into four zones, reframing the loop between zones Microbiology section in KAMC: Instruments used in microbiology section: 19 | P a g e instrument information Bactec 9240 -Used for blood culture. - aid in diagnosis of bacteremia. - blood bottles are used as a container for blood samples. Blood culture bottles -There are two type pf bottles: AEROBIC (blue cover) and anaerobic (purple cover) -It contains: 1-SPS (sodium polyantha sulfonate) that act as anticoagulant, prevent phagocytosis and complement reaction (liquid part) 2- a sensor (the gel on the bottom) to detect bacterial growth that secret co2 during multiplication it gives alarm when growth detected. 3-Resin or beads: acts as neutralization and absorption of antibiotic. The amount must be from 8-10ml for adult and 1-5 ml for children. Microscan walkaway 96 -Aid in identifications of bacteria and play an essential role for antibiotic sensitivity test (AST). -Principle: colorimetric it detects the change in color due to different biochemical reaction related to specific bacteria. 20 | P a g e pictures Vitek 2 compact Component of vitek -Aid in identifications of bacteria and play an essential role for antibiotic sensitivity test (AST). -Principle: colorimetric it detects the change in color due to different biochemical reaction related to specific bacteria. -an identification cart (kit) used for bacterial identification whether gram positive or negative or fungi - each kit used for specific organisms. -It contains all biochemical reaction according to isolated bacteria. - other kit used for (AST). Rack used for kits General purpose incubator. 21 | P a g e -for fungal, bacterial growth -each incubator has specific temperature for growth depending on the type of organism. Bacteria growth in>> 36c while fungal in>> 25c -The duration of bacterial growth takes from 24h to 48h -Fungal from 1 week to 4 weeks Used for culture purpose to insure the safety for the specialist and removing any contamination from the air. Class II biohazard safety cabinet. Some bacteria need CO2 for Auto-flow IR water jacketed CO2 incubator. growth so this type of incubator provides the suitable amount of CO2 to each bacterium. Microbiology divided into five sections: 1-reception and receiving of clinical samples 2-respiratory bench 3-blood & body fluid& tissue bench 4-urine and stool bench 5-mycology bench First: reception and receiving area This area receives different type of clinical samples include: 1-Urine 2- stool 3- blood 4- tissues 5- sputum 6-BAL 7-rectal swab 8- throat and mouth swab 9- eye and ear swab 10- wound swab. How do we receive samples? According to acceptance and rejection criteria which is: 22 | P a g e Rejection criteria: 1-All unlabeled samples must not receive. 2-all miss labeled samples must be verified before receiving. 3-old samples must be rejected. 4-unseffecient mount of samples must not receive and send a request to gather another sample. 5-unsterile container. 6-if there is a leakage of samples inside the biohazard bag. 7- if the sample is sputum and there is a saliva more than the accepted we must rejected because it contains an epithelial cell that interfere with the result. 8- tissue samples inside a formaldehyde container because, the formaldehyde will kill any present bacteria and we need it for the diagnosis. 9- needle with an aspiration samples, recap of needle is not allowed for safety purpose. Which type of media used with the different samples: CULTURE specimen Ear Nasal secretion Throat Mouth sputum BAL BA aerobic BA anaerobic 23 | P a g e MAC CHOC SAB * * * * * Direct examination BA\MAC Thioglcate broth * Gram stain Wet preparation KOH * * eye Skin tissue Catheter tip Wound swab Pus aspirate Body fluid CSF urine HVS MRSA VRE stool * * * * * *3 * * * * * Blood and mannitol salt agar with oxacillin Blood and bile esculin agar XLD and selenite broth Key: (*) when fungal infection is suspected, (*3) when yeast cells seen in urine examination. Note: Each day you must check the quality control of each machine including, biohazard class II safety cabinet, microscopes, stains, general purpose incubator, refrigerator temperature and media stability. Second: respiratory bench In this area, we must know the most common pathological bacteria present in respiratory tract and these bacteria classified according, the site of infection to: 24 | P a g e * Upper and lower tract infection: Nose: Mouth: staphylococcus spp yeast Throat: Nasopharyngeal: streptococcus. pyrogen mortadella spp Lower tract includes: -H. influenza -Moraxella -s. pneumonia Morphological characteristics of bacteria on agar: -H. Influenza is growing on chocolate agar. Notice semiopaque, gray-white, mucoid. -Moraxella -Gram negative- short rods coccobacilli- cocci -Gram negative- small- cocci -Colonies appear as Smooth with a grayish Wight color. -Oxidase + catalase+ Aerobic -When the colony pushed with loop they scoot a cross the media. -Oxidase + catalase+ 25 | P a g e -strep. pneumonia -Gram positive- cocci with alpha hemolysis. -Colonies appear as mucoid green in color. -bile solubility test + -strep. pyrogen Gram +ve cocci arranged in chains -staph. aureus Gram +ve cocci arranged in clusters -Aerobic, non-motile and produce grey-white colonies. - coagulase positive - Pin point colonyies with Wide zone of hemolysis. -yeast appears like star in shape Wight off in color. -Aerobic, non-motile, produce golden yellow colonies - -haemolytic Significant and insignificant growth of bacteria: According, to the number of organism on the streak if: 1-Low number of organism grow in one streak considered as normal flora or insignificant growth. > the result is normal 2-moderat number of growth in media there are three cases: - If one type of organism grow considered a pathogen and we must use the Vitec machine for identification. -If two type of organisms grow on media (the many in number) > pathogen and (low) > insignificant. -If three type pf organisms grow considered as mixed and we must reject the sample> no farther work out. Screening test: -MRSA screening considered an important test for inpatient to monitor their health. -MRSA grow on mannitol salt agar as we mentioned before. 26 | P a g e Gastrointestinal (GTT) test: -For yeast identification -sample used (serum or plasma) Procedure: 1-apply the wet mount technique by taken a small sample from the agar an place it on microscope slid then cover it with a cover slip. 2-see it under 40X power 3-if the organism appears like a pudding shape perform the GTT test Pudding shape 4-take enough colony and add it into tube contains serum. 5-incubate the sample in general incubator for 2-3 hours. 6-see the result after perform the wet mount on 40X. Result: Candida Albicans germ tube -If other organism is different from the germ tube right (other than candida albicans) 27 | P a g e Third: blood bench area When the blood cultivation is needed? In case of: - High temperature of unknown cause - Endocarditis which caused by inappropriate tooth extraction and the causative organism will be s. pyridines -No antibiotic administration -The amount of sample (blood) that will be collected from adult must be from 8-10ml -While children the mount arrange between 1-5 ml -Blood are collected in special container called (blood culture bottles) as we mentioned before. -after bottles monitoring they placed in BACTEC machine for processing and identification of the positive organism. -The duration of result takes about 5 days if there is no alarm sound for positive result we considered it as negative (normal sample). Steps to deal with the positive results If the positive result was in Aerobic bottles take it and do the culture process: If the positive result was in Anaerobic bottles take it and do the culture process: -blood agar -blood agar O2 -chocolate agar -blood agar CO2 MacConkey's agar -chocolate agar -gram stain MacConkey's agar Note: All the samples must be sterile 28 | P a g e -gram stain Bacterial present in CSF samples according, to the age Children: H. influenza type B Newborn: Adult and elderly: S. agalactia -s. pneumonia E. coli - Neisseria - nectria monocytogen How can we differentiate between bacterial and viral infection? By three ways: 1-gram staining technique. 2-cell count (bacterial infection indicate a high number of neutrophils, viral infection indicates a high number of lymphocyte count, TB infection both will be high). 3-glucose concentration (bacteria will consume it so the amount will be low, in viral infection glue> normal. Forth: urine and stool bench In this area, we are looking for family of Enterobacteriaceae I & II. General characteristic's: 1-Found in the GIT of humans and animals. 2-Gram negative, non-spore forming. 3- Aerobic and facultative anaerobes. 4-Grow in blood and MacConkey's agar. 5-Ferment glucose and produce acid. 29 | P a g e Enterobacteriaceae I (lactose fermenting): -Escherichia -Klebsiella -Enterobacter -Citrobacter Enterobacteriaceae II (non-lactose fermenting): -Salmonella -Morganella -Shigella -Proteus - Serratia Infection by Enterobactericeae: MacConkey's agar plate Morphology of most common bacteria: 1-E. coli: is part of normal flora of intestine of humans and animals. 30 | P a g e -Aerobic, Gram-negative bacilli. -Produce pink colonies (by fermenting lactose) on MacConkey agar. -Motile, non-spore formers. (characterized by pink shadow) 2- Klebsiella: -Aerobic, Gram-negative bacilli. -Produce large, mucoid colonies on blood and MacConkey agar and ferments lactose. -Non-motile, non-spore formers. -Urease +ve after 18-24 hours. klebsiella on MacConkey agar klebsiella on blood agar 3-salmonellae: are motile, aerobic, gram-negative bacilli. -Do not ferment lactose. -Produce H2S from Sulphur-containing amino acids. -Grows on XLD agar and produce black colony 31 | P a g e E. coli on MacConkey agar 4- Shigella: -non-motile, aerobic, gram-negative bacilli. -Do not ferment lactose. -Do not produce gas during carbohydrate fermentation. - appear as pink to red colony on XLD agar. 5-proteus: - Produce Swarming growth on blood agar. -NLF colonies on MA, DCA and SS Agar. Swarming is inhibited on these media due to presence of bile salts. Some immportant NLF bacteria: Pseudomonas: 1-G –ve rods, strict aerobes, and motile. Non-fermenter, Oxidase positive. 2-Fruit smell and produce green pigment on MacConkey and blood agar due to: Thiocyanide day and pyoverdine day production Pseudomonas on MacConkey agar 32 | P a g e Pseudomonas on blood agar Acinetobacter: 1-G –ve rods, nonmotile. Nonfermenter, Oxidase negative. 2-regular circle colony Acinetobacter on MacConkey agar Classification and testing of Antibiotics: Antimicrobial agents can be classified according to their mode of activity: -bacteriostatic: inhibit bacterial growth – bactericidal: kill bacteria Classes of antibiotics: Cell wall synthesis inhibitors Beta-lactams antibiotics include: 1-cephalosporins include: protein synthesis inhibitors 1-Aminoglycosides include: -Streptomycin -cefepime 2- penicillins include: - Penicillin p Ampicillin Oxacillin Pipracillin Other beta lactams (imipenem& meropenem &aztreonam) 1-polymyxins: 1-Sulphonamides include: -Sulfamethoxazole -Amikacin -cefotaxime -ceftazidime Nucleic acid synthesis inhibitors -Trimethoprim -Gentamicin 2- phenicol's include: - Chloramphenic ol 3-Tetracycliness include: - Tetracycline Doxycycline 4-Macrolides include: - Erythromycin Azithromycin 2- Quinolones (Nalidixic)or Fluoroquinolones include: - 1-Levofloxacin 2-Norfloxacin 3-Ciprofloxacin 3-Rifamicins include: - Rifampicin 4-Nitroimidazoles include: - 33 | P a g e Cell membrane synthesis inhibitors Metronidazole -polymyxins E (colistin) -polymyxin B Notes: Cell wall synthesis: -penicillins are a large group of antibiotics that include Aminopenicillins (ampicillin& amoxicillin) penicillinase-resistant penicillin's (oxacillin), penicillins with betalactamase inhibitors (amoxicillin\clavulanic acid). -methicillin is an antibiotic that was originally used to treat Gram-positive infections but is no longer available for clinical use. It was replaced by other agents (oxacillin). -cephalosporin's are further classified into 1st,2nd,3rd and 4th generation according to their antimicrobial properties. -cefoxitin is a cephalosporin-like antibiotic that is used for the screening of MRSA. Protein synthesis inhibitors (PSI): Other PSI compounds include: linezolid and quinupristin-dalfopristin, both of which are considered treatment options for infections caused by VRSA. Cell membrane synthesis inhibitors: These compounds are highly toxic and poorly absorbed therefor, they are usually used topically and mainly indicated for Gram-negative infections. Anti-TB drugs RNA synthesis inhibitors include: -Rifampicin -Ethambutol(EMB) Mycolic acid synthesis inhibitors include: -Isoniazid(INH) Fatty acids synthesis inhibitors include: -pyrazinamide(PZA) Notes: treatment of TB is usually a combination of: 1-rifampicin, INH, PZA and EMB first for the first 2 months (intensive phase) 34 | P a g e 2-rifampicin and INH for the further 4 months (continuation phase) 3-in case of TB resistance, other antibiotics are used such as aminoglycosides(streptomycin)and fluoroquinolones e.g.(moxifloxacin) Antimicrobial susceptibility testing (AST) Disc susceptibility test (Kirby Bauer method) Zone of inhibition=diameter(mm) Antibiotic disc Intermediate (I) Mueller Hinton agar MH Bacterial growth Sensitive(S) Resistant ® Inoculate the plate with 0.5 McFarland bacterial suspension + Add antibiotic discs incubate (overnight) > bacterial growth. Interpret reading using standard chart: Sensitive>drug is likely to work on patient. Intermediate>drug may work on patient in higher doses OR in combination with another drug. Resistant> drug will not work on patient. Note: McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range to standardize microbial testing. An example of such testing is antibiotic susceptibility 35 | P a g e testing by measurement of minimum inhibitory concentration which is routinely used in medical microbiology and research. If a suspension used is too heavy or too dilute, an erroneous result (either falsely resistant or falsely susceptible) for any given anti-microbial agent could occur Minimum inhibitory concentration (MIC) -Is the lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism. -MBC (minimum bacterial concentration) is the lowest concentration of an antimicrobial agent that kill a microorganism. -MIC and MBC are measured in micro gram\ml or mg\l -bactericidal drugs have MBC values close to their MIC. -bacteriostatic drugs have MBC values much higher than their MIC values. Broth dilution method: - 36 | P a g e E Test Method: Place E test strip on plate inoculated with 0.5 McFarland bacterial suspension Incubate (overnight) Bacterial growth Zone of inhibition Mueller Hinton agar E strip Example: MIC= 0.25 mg\l point of zone and strip interception Note: 0.5MacFarland =a.5×10^8 CFU \ml 37 | P a g e Report MIC value using standard chart as: S, I OR R )صل الل م سلم على سيدنا مح د على آله صحبه ج عين ) ِ .م اصبت ف بتوفيق من ه إ خطأ ف ن نفسي قل حيلتي Remember: Be a great wherever you go, and be proud of yourself and what you are accomplished so far. Always remember (great things take time). GOOD LUCK 38 | P a g e