Practical Microbiology Lab 1 PDF

Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 1 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــ...

Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 1 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Orientation to The Microbiology Laboratory Microbiology is the science that study of microorganisms (MO.) and including bacteriology, mycology, virology, and parasitology. Microbiology lab. is a place to grow and study tiny organisms, called microorganisms, and these Microbes can include bacteria, fungus, virus , and parasite. Bacteria Fungi Safety Procedures and Precautions  Hands and bench tops are kept clean with disinfectants,  laboratory coats are worn (wore), long hair is tied back,  working areas are kept clear of all unnecessary items,  containers used for specimen collection or culture material are pre- sterilized and capped to prevent entry by unsterile air,  nothing is placed in the mouth, Personal conduct in a microbiology laboratory should always be quiet and orderly,  the instructor should be consulted promptly whenever problems arise,  any student with a fresh unhealed cut, scratch, burn, or other injuries should notify the instructor before beginning or continuing with the 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 1 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ laboratory work.  1.Always read the assigned laboratory material before thestart of the laboratory period. 2.To be admitted to the laboratory, each student should wear clean,knee-length laboratory coat. 3.At the START and END of each laboratory session, students shouldclean their assigned bench-top area with a disinfectant solution provided. 4. Learn good personal habits from the beginning:  Tie back long hair neatly away from the shoulders.  Do not wear jewelry to laboratory sessions.  Keep fingers, pencils, and such objects out of your mouth.  Do not smoke, eat, or drink in the laboratory.  Do not lick labels with your tongue.  Do not wander about the laboratory due to unnecessary activitycan cause accidents, and promote contamination. 5. Before leaving the laboratory, carefully wash and disinfect yourhands. 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Sterilization & Disinfection Sterilization: is a process that kills all forms of microbial life, including bacterial spores, which are highly resistant. Disinfection: is a process that destroys pathogenic organisms, but not necessarilyall microorganisms or spores. [Chlorine, formaldehyde]. Antiseptics: less toxic materials used to kill microorganisms on the surface of skin and mucous membranes [such as iodin and ethanol]. Chemotherapy: Chemicals used internally to kill or inhibit growth of microorganisms within host tissues. Methods of Sterilization 1- Chemical Method Chemicals vary greatly in their ability to kill microorganisms by one of thethree mechanisms: a. disruption of the cell membrane [ex: alcohol, detergent, phenol] b. modification of proteins [ex: chlorin, iodin, heavy meatal, hydrogenperoxide, formaldehyde] c. modification of DNA [ex: crystal violet, Malachite green] 2- Physical Method a. Heat 1- Dry heat 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬  Flaming: Used for Loop, inoculating wires, glassware, etc...  Incineration: Disposal of infectious materials  Oven:  180°C for two hours  used for glassware, metal wares, oils, powders; etc... 2- Moist heat  Boiling:  100°C for 30 minutes.  Kills everything except bacterial spores  Autoclaving  15 atm pressure, 121°C and for 15min  sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things.  Pasteurization  don't use boiling temperature because it cause protein denaturation.  Methods: Slow procedure: milk is pasteurized for 30 minutesat about 62°C. Rapid procedure: milk is pasteurized for 15 minutes at about 72°C. b. Radiation  ultraviolet (UV) light: used to sterilize workspaces and tools 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ used inmicrobiology laboratories  X-rays: used in medicine such as sutures and surgical gloves, andplastic items, such as syringes. c. Filtration:  is the preferred method of sterilizing certain solutions  filter is composed of nitrocellulose and has a pore size of 0.22 um. Figure : Microbial control method. Factors influencing on the effectiveness of antimicrobial agents 1. The number of microbes: larger population requires a longer time to die than asmaller one. 2. Microbial characteristics: Bacterial endospores are much more resistant to mostantimicrobial agents than are vegetative forms. 3 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 3. Time of exposure: Chemical antimicrobials often require extended exposure toaffect more-resistant microbes or endospores. 4. Environmental influences: Most disinfectants work somewhat better in warmsolutions. 5. Concentration or intensity of an antimicrobial agent: Table : Clinical use of Disinfectant and Sterilization. Methods Mechanism of Action Comment Preferred Use Heat Protein denaturation Kills vegetative bacterial and fungal Dishes, basins, pitchers, 1. Moist heat pathogens and almost all viruses various equipment a. Boiling within 10 min; less effective on endospores. b. Autoclaving Protein denaturation Very effective method of Microbiological media, sterilization; at about 15 psi of solutions, dressings, pressure (121°C), all vegetative equipment, and other cells and their endospores are items that can killed in about 15 min. withstand temperature and pressure c. Pasteurization Protein denaturation Heat treatment for milk (72°C for Milk, cream about 15 sec) that kills all pathogens and most nonpathogens. 2. Dry heat Burning contaminants Very effective method of Inoculating loops a. Flaming to ashes sterilization. b. Incineration Burning to ashes Very effective method of Biohazard wastes sterilization. c. Hot-air [oven] Oxidation Very effective method of Empty glassware, sterilization but requires instruments, needles, temperature of 170°C for about 2 and glass syringes hr. Filtration Separation of bacteria Removes microbes by passage of a Useful for sterilizing from suspending liquid or gas through a screenlike liquids (e.g., enzymes, liquid material; most filters in use consist vaccines) that are of cellulose acetate or destroyed by heat nitrocellulose. Radiation Destruction of DNA Not widespread in routine Sterilizing 1. Ionizing sterilization. pharmaceuticals and [gamma ray] medical and dental supplies 2. Nonionizing Damage to DNA Radiation is not very penetrating. Control of closed [UV] environment with UV lamp 4 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 3 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Culture media Culture Medium: a special medium used in microbiological laboratories for the growth, isolation and identification of microorganisms. A culture medium is composed of different nutrients (carbohydrate, lipids, amino acids, vitamins as well as inorganic compounds) Types of Culture Media Media can be classified according to three properties 1. Physical state 2. Chemical composition, and 3. Functional type (purpose). Table : Three Categories of Media Classification Physical State Chemical Composition Functional Type 1-Liquid 1- Chemically defined 1-General purpose (synthetic) 2-Semiliquid 2- Complex; not chemically 2-Enriched defined 3-Solid 3-Selective 4-Differential 5-Transport 6-Enumeration 7-Assay 8-Anaerobic growth Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Physical States of Media 1- Liquid media  media without any agar or gelatin  Commonly used for:  General culture  Biochemical test  Susceptibility test 2- Semisolid Media  Contain small amount of solidifying agent (agar or gelatin)  Media with 0.5 or less of agar  Commonly used for:  Microaerophilic culture  Motility test 3- Solid media  Contain 1.5 % – 2 % agar  Commonly used for  Agar plate  Isolation of bacterial colonies 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Chemical composition of Media 1- Chemically defined [synthetic] media  Media whose exact chemical compositions are known are termed defined (also known as synthetic)  contain essential compounds such as salts and amino acids dissolved in water, or may be composed of a variety of defined organic and inorganic chemicals. Example: Mineral glucose medium 2- No Chemically defined [nonsynthetic] media  If even one component of a given medium is not chemically definable,  Complex media contain extracts of animals, plants, or yeasts, examplesare blood, serum, and meat extracts. Functional Type 1- General purpose  support the growth of a variety of microbial life  Also now nutrient media  Examples  nutrient agar and broth,  brain-heart infusion, and  trypticase soy agar (TSA) 2- Enriched  Support the growth of *fastidious bacteria  contains complex organic substances such as blood, serum, 3 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ hemoglobin, or special growth factors (specific vitamins, aminoacids)  Examples  Blood agar  Chocolate agar *Bacteria that require growth factors and complex nutrients aretermed fastidious. 3- Selective media  Allow the growth of desired bacteria while inhibitory other types  Contain inhibitory agents like antibiotic, bile salts,  Examples :  MacConkey agar [selective for gram negative]  Mannitol salt agar (MSA) contains a high concentration ofNaCl (7.5%) selective for Staphylococcus.  Colistin-nalidixic agar [selective for gram positive] A-Mannitol Salt Agar B- MacConkey agar C- MacConkey agar 4 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 2 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ A-Klebsiella on MacConkey agar B- E. coli on MacConkey agar Table : Selective Media, Agents, and Functions Medium Selective Agent Used For MacConkey agar Bile, crystal violet Isolation of gram-negative enterics Salmonella/Shigella Bile, citrate, brilliant Isolation of Salmonella and Shigella (SS) green Lowenstein-Jensen Malachite green dye Isolation and maintenance of Mycobacterium Mannitol salt agar Sodium chloride [NaCl] Isolation of Staphylococcus species 5 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 4 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 1- Differential media  allow multiple bacteria to grow but with distinguishable colonialcharacteristic.  Allow for preliminary characterization of bacteria.  Example:  Blood agar: is a type of enriched medium but give hemolyticcharacteristic of bacteria  Combination media [selective and differential media]  Mannitol salt agar selective and differentiation ofStaphylococci. 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 4 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Table : Differential Media Medium Substances That Differentiates Between Facilitate Differentiation Blood agar Intact red blood cells Types of hemolysis displayed by different species of Streptococcus Mannitol salt Mannitol, phenol red Species of Staphylococcus agar MacConkey agar Lactose, neutral red Bacteria that ferment lactose (lowering the pH) from those that do not Urea broth Urea, phenol red Bacteria that hydrolyze urea to ammonia Sulfur indole Thiosulfate, iron H2S gas producers from nonproducers motility (SIM) Triple-sugar iron Triple sugars, iron, and Fermentation of sugars, H2S production agar (TSIA) phenol red dye 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 4 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 1- Transport media:  Used when specimen cannot be processed immediately.  Preserve the viability of microorganism in specimen but not allowto growth.  Most transport media contain a salt and buffer to preventdestruction of cell by enzyme. 3 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 4 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 1- Enumeration media:  Used to count the numbers of organisms in milk, water, food, soil,and other samples 2- Assay media:  Used to test the effectiveness of antimicrobial drugs on the growthof microorganisms 3- Reducing medium:  Contains a substance (thioglycolic acid or cystine) that absorbsoxygen or slows the penetration of oxygen in a medium,  Important for growing anaerobic bacteria. 4 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Media Preparation Tool, Glassware and Instruments required for media preparation: 1- Conical flask 2- Cylinder 3- Petri dish 4- Cotton or Aluminum foil5- Filter paper 6- Bunsen burner 7- Electronic balance 8- Autoclave 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Samples collection and Culturing Common Specimens 1- Blood 2- Urine 3- Sputum 4- Swab [Throat, Nasal, Ear, Eye, HVS] 5- Stool 6- Pus 7- CSF [cerebrospinal fluid]  Specimens must be collected correctly  If not my not grow in culture  Contamination may be mistakenly identified  Patient may be received incorrect or harmful therapy Specimen collection [Guidelines]  Avoid causing harm or discomfort to patient  Collect from appropriate site  Obtain specimen at correct time  Use appropriate devices  Obtain sufficient quantity of specimen  Obtain specimen prior to the start of antimicrobial therapy  Label correctly 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ General Equipment’s required What containers to used?  Container must be leak proof  Unbreakable  For cultures sterile container a must 1- Blood collection [for culture]  Disinfect venipuncture site with 70% alcohol  Improper handling of syringes increaseschances of contamination. Handling and transport  Collect into blood culture bottles [broth]  Request must contain relevant patient information  Sent immediately to the laboratory with request 3 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 2- Urine Specimen  Clean-voided midstream Containers  Sterile, screw-cap container  From babies, urine is collected in sterileplastic bags Patient preparation  Clean area with soap and water, then rinsewith water; after several mL have passed, collect midstream of urine Transport  Transport to laboratory within one hour or  Kept at 4 C to avoid multiplication of bacteria in urine. Culture  Blood agar  MacConkey agar 3- Sputum Collection of Specimen  Patient is instated to take a deep breath and cough up 4 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ sputumdirectly into a wide mouth sterile container  Avoid saliva or postnasal discharge  Minimum volume 1 ml. 4- Swab a) Throat swab  Aplain cotton wood swab should be used to collect as much exudate as possible from tonsils. Posterior pharyngeal wall and other area that is inflamed or bears exudates.  Swab posterior pharynx and tonsils; routine culture for group A Streptococcus (S. pyogenes) only b) Nasopharyngeal swab  Tilt Head backwards.  Insert flexible swab through nose into posterior nasopharynx and rotate for 5 seconds; specimen of choice for Bordetella pertussis. c) Ear swab  No drops should have been used 3 hours prior to takingthe swab.  Place the swab into the outer ear and rotate gently once. 5 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 5 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ d) Pus swab {from wound}  After careful cleaning of the wound site a swab was taken from the innerside of the wound or the area where pus is accumulated.  Rotate the swab gently once. 6 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 6 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 5- Stool Sample  Clean, leak-proof container; transfer feces to enteric transport medium (Cary-Blair) if transport will exceed 1 hr  Transfer container quickly to the laboratory 6- Pus Sample  Aspirate from the abscess or the wound in sterile container  Should be transported immediately to the laboratory. 7- CSF [ Cerebrospinal Fluid] Collection is done:  By lumbar puncture  In sterile tubes  Under aseptic condition  By trained physician only  Sterile screw cupped container to be used Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 6 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Aseptic Technique The most important tool for transferring cultures is the wire inoculatingneedle or loop. It can be quickly sterilized by heating it to red hot in a Bunsen burnerflame. A dry needle may be sterilized by holding it at a 30-degree angle in theouter part of the flame. Always flame the loop immediately before and after use! Allow it to cool before picking up an inoculum of bacteria (or you will kill the bacteria Isolation Methods of Microorganisms Isolation is done by: 1-streaking plate method. 2-spreading plate method 3-puoring plate method Streaking plate method Purpose of streaking  To isolate single colonies of bacteria on an agar plate. Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 6 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬  This is useful when we need to separate single colonies of bacteria from a mixed culture (more than one species)  or when we need to study the colony morphology of bacteria.  To identify the organism: biochemical tests to identify bacteria are only valid when performed on pure cultures. Procedure 1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool. 2. Remove a small amount of bacteria growth [either a loopful from a broth culture or a single colony from plate or slant] with the sterile inoculating loop. 3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back and forth motion (see area 1 in the figure 1). 4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the second quarter of the plate (area 2). 5. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 2), extend the streaks into the third quarter of the plate (area 3). 6. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 3), extend the streaks into the center fourth of the plate (area 4). 7. Flame your loop once more. Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 6 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Figure (1): streak-plate technique. Results The streaked plate is incubated at 37°C for 24 hours. Examine the colonies grown in the plate carefully. All colonies should have the same general appearance. If there is more than one type of colony, each type should be streakedagain on a separate plate to obtain a pure culture. Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 6 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Slant: Slanting gives the bacteria a greater surface area on which to grow in a tube. Agar slants are also useful in maintaining bacterial cultures, more so than stacks of Petri dishes. Bacteria are inoculated onto a slant using a loop and grow in the surface of the agar. Slant/Deep: Similar to a slant but creates a deep zone, commonly called the ‘butt’. It gives the ability to grow bacteria in both an aerobic (surface of the slant) and an anaerobic (butt of slant). Slant/deeps are inoculated by stabbing a needle into the butt and then immediately streaking across the surface of the slant. Deep: This type of culture is used for the growth of anaerobic bacteria which grow in the absence of oxygen and are inoculated by stabbing the media with a needle. Broth: Allows for the growth of large volumes of bacteria, the level of growth can be assessed based on the turbidity of the culture. Bacteria are inoculated into a broth using a loop. Broth+Durham Tube: in which an upside-down smaller tube, called a Durham tube, is placed. Durham tubes are used to detect the production of gases, such as CO2 or N2, by microorganisms. The tube is initially filled with the medium and then collects gas as the bacteria grow, creating a bubble Bacteria are inoculated into a broth+Durham tube using a loop. Plate: Plates are particularly helpful in isolating a specific species of bacteria, which is not possible in a liquid medium Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 7 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Bacterial Staining Purpose of staining Q: Why do we stain microorganisms? Bacteria are microscopic and colorless organisms. 1- In order to visualize them and study their structure and shape, to make them more easily visible. 2- Staining organisms enable us to observe different microbial characteristics. Shape and arrangement Gram reactions Endospore production Capsule Production Pathogenic bacteria Q/ What is the stain? Stains or dyes are chemicals with two parts: 1 -Chromophore: the part that has the color. 2 -Auxochrome: the part that helps in the coloring properties. The chromophore of a stain may be charged either +ve charge or -ve charge. 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 7 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Types of stains 1- Basic stain: A positively charged (cationic dye): Chromophore is a positive ion dye attracted by the bacteria so the cells of bacteria stained e.g. Crystal violet, methylene blue, safranine Bactria 2- Acidic stain: A negatively charged (an anionic dye) 1- Chromophore is a negative ion dye rejected by the cell and background of slid stained, bacteria are colorless. e.g. Eosin, India ink Bacteria Note Bacteria are slightly negative, so are attracted to positive chromophoreof basic stain. 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 7 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Types of Staining techniques 1- Simple staining: Use of single stain For visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads) 2- Differential staining: Use of two contrasting stains. Separation into groups e.g. 1-Gram stain 2-Acid-fast stain Visualization of structures e.g. 1- Flagella stain 2- Capsule stain 3-Spore stain 4-Nuclear stain The first step in staining 1- Smear Preparation --- is always the first step in any microbial stain. It is spreading microbial cells on a clean glass slide and allowing it to air dry and then heat fix. Benefit of Heat Fixation 1. Kills the organism. 2. It causes the organisms to adhere to the slide. 3. It alters the organisms so that they more readily accept stains. 3 ‫‪Assist. Prof. Dr. Ameer Mezher Hadi‬‬ ‫‪Al-Zahrawi University College‬‬ ‫‪Lab. 6‬‬ ‫‪Practical Microbiology‬‬ ‫‪Department of Dentisy‬‬ ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫‪Figure (1): Procedure of smear preparation‬‬ ‫‪4‬‬ ‫‪Assist. Prof. Dr. Ameer Mezher Hadi‬‬ ‫‪Al-Zahrawi University College‬‬ ‫‪Lab. 6‬‬ ‫‪Practical Microbiology‬‬ ‫‪Department of Dentisy‬‬ ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫‪Figure (2): Slide Smear.‬‬ ‫‪5‬‬  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Colony Morphology and Cultural Characteristics Microbial Colony: is defined as a visible cluster or mass of microorganisms growing on the surface of or within a solid medium, originating from a single cell. Colony morphology: is a method that scientists use to describe the characteristics of an individual colony of bacteria growing on agar in a Petri dish. It can be used to help to identify them. Colony Morphology A / On solid medium 1- Shape 2- Elevation3- Size 4- Margin 5- Surface 6- Density 7- Color 8-Pigment9- Odor 1  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 1-Shape of the colony Punctiform Circular Irregular Filamentous Rhizoid 2- Elevation of the colony This describes the (side view) of a colony. raised, convex, flat, umbilicate (depressed center, concave) , umbonate (raised or bulging center, convex). “Diphtheroid” colonies with dry appearance and Small, umbonate center colony of Eikenella umbonate center growing on sheep blood agar 2 corrodens on chocolate agar. (SBA) plate  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 3-Size 1- large, 2- medium, 3-small, or 4-pinpoint. Left, (SBA) plate: small, white colonies are gram- positive cocci. Right, SBA: large, gray, mucoid colonies are enteric gram-negative rods 4-Margins of the colony 1- smooth, 2- filamentous, 3- wavy (undulate) 4- irregular 5- concentric Swarming colonies of Proteus spp. Irregular 3  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 5- Surface of the colony 1- Smooth 2- glistening 3- rough 4- wrinkled 5- dry 6- Mucoid Rough Glistening Wrinkled Mucoid Dry 4  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 6-Density 1- transparent (colonies allow light to pass through the colony) 2- translucent (colonies allow some light to pass through the colony) 3- opaque (light not pass through the colony) 7-Color white (Coagulase-negative staphylococci) gray (Enterococcus spp.) yellow (Neisseria spp. (nonpathogenic) or off white) buff (Diphtheroids) white pink Gray red black 5  Assist. Prof. Dr. Ameer Mezher Hadi Al- Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫ـــــــــــــــــــــ‬ 8- Pigment Pigment production is an inherent characteristic of a specific organism confined generally to the colony, although some pigments will diffuse through the culture medium. Pigment (green colour) on nutrient agar (P. aeruginosa ) 9- Odour ▶ Microorganisms can produce different types of volatile compounds whichmay give characteristics smell, pleasant scent or pungent odor. ▶ Production of these volatile chemicals depends on the metaboliccharacteristics of that particular organism. Unpleasant smell: When an anaerobic 6  Assist. Prof. Dr. Ameer Mezher Hadi Al- Zahrawi University College Lab. 8 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫ـــــــــــــــــــــ‬ infection is suspected,specimen is often foul- smelling. Rotten cooked fishy odor: Proteus mirabilis Sweet grape-like scent: Pseudomonas aeruginosa Caramel odor: Streptococcus milleri 7  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 9 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Staphylococcus Staphylococci are spherical gram-positive cocci arranged in irregular grapelike clusters.All staphylococci produce catalase, whereas no streptococci do (catalase degrades H2O2 into O2 and H2O). Catalase is an important virulence factor. Bacteria that make catalase can survive the killing effect of H2O2 within neutrophils. Three species of staphylococci are important human pathogens: S. aureus, S. epidermidis, and S. saprophyticus of these three, S. aureus is by far the most common and causes the most serious infections. Staphylococcus aureus is distinguished from the others primarily by coagulase production.Coagulase is an enzyme that causes plasma to clot by activating prothrombin to form thrombin. Thrombin then catalyzes the activation of fibrinogen to form the fibrin clot. Staphylococcus epidermidis and S. saprophyticus are often referred to as coagulase-negative staphylococci. Figure - 1: Staphylococcus aureus—Gram stain. Arrows point to two “grapelike” clusters of gram-positive cocci. 1  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 9 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Table: Staphylococci of Medical Importance Species Coagulase Typical Important Typical disease Production hemolysis Features S. aureus + β Protein A on Abscess, food surface poisoning, toxic shock syndrome S. epidermidis - None Sensitive to Infection of prosthetic heart novobiocin valves and hips; common member of skin flora S. saprophyticus - None Resistant to Urinary tract novobiocin Staphylococcus aureus produces a carotenoid pigment called staphyloxanthin, which imparts a golden color to its colonies. This pigment enhances the pathogenicity of the organism by inactivating the microbicidal effect of superoxides and other reactive oxygen species within neutrophils. Staphylococcus epidermidis does not synthesize this pigment and produces white colonies. The virulence of S. epidermidis is significantly less than that of S. aureus. Two other characteristics further distinguish these species, namely, S. aureus usually ferments mannitol and hemolyzes red blood cells, whereas S. epidermidis and S. saprophyticus Coagulase test—Upper tube inoculated with Staphylococcus aureus; lower tube inoculated with Staphylococcus epidermidis. Arrow points to clotted plasma formed by coagulase produced by S. aureus. 2  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 9 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Hemolysis of red cells by hemolysins produced by S. aureus is the source of iron required for growth of the organism. The iron in hemoglobin is recovered by the bacteria and utilized in the synthesis of cytochrome enzymes used to produce energy. Figure-2: Coagulase test—Upper tube inoculated with Staphylococcus aureus; lower tube inoculated with Staphylococcus epidermidis. Arrow points to clotted plasma formed by coagulase produced by S. aureus. Laboratory Diagnosis 1-Smears from staphylococcal lesions reveal gram-positive cocci in grapelike clusters 2-Cultures of S. aureus typically yield golden-yellow colonies that are usually β- hemolytic. Staphylococcus aureus is coagulase positive. 3  Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 9 Practical Microbiology Department of Dentisy ‫ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 3-Mannitol-salt agar is a commonly used screening device for S. aureus. Staphylococcus aureus ferments mannitol, which lowers the pH causing the agar to turn yellow whereas S. epidermidis does not ferment mannitol and the agar remains pink. 4-Cultures of coagulase-negative staphylococci typically yield white colonies that are non hemolytic. 5-There are no serologic or skin tests used for the diagnosis of any acute staphylococcal infection. 6-In toxic shock syndrome, isolation of S. aureus is not required to make a diagnosis as long as the clinical criteria are met. Laboratory findings that support a diagnosis of toxic shock syndrome include the isolation of a TSST producing strain of S. aureus and development of antibodies to the toxin during convalescence, although the latter is not useful for diagnosis during the acute disease. Treatment 1- Treatment with a combination of a β-lactamase–sensitive penicillin (e.g., amoxicillin) and a β-lactamase inhibitor (e.g., clavulanic acid) is also useful. 2-The drug of choice for these staphylococci is vancomycin, to which gentamicin is sometimes added. Daptomycin is also useful. 3-Mupirocin is very effective as a topical antibiotic in skin infections caused by S. aureus. It has also been used to reduce nasal carriage of the organism in hospital personnel and in patients with recurrent staphylococcal infections. 4-Cefazolin is often used perioperatively to prevent staphylococcal surgical-wound infections. 4 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 13 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ LISTERIA MONOCYTOGENES Listeria monocytogenes causes meningitis and sepsis in newborns, pregnant women, and immunosuppressed adults. It also causes outbreaks of febrile gastroenteritis. It is a major cause of concern for the food industry. Laboratory Diagnosis Laboratory diagnosis is made primarily by Gram stain and culture. The appearance of gram-positive rods resembling diphtheroids and the formation of small, gray colonies with a narrow zone of β-hemolysis on a blood agar plate suggest the presence of Listeria. The isolation of Listeria is confirmed by the presence of motile organisms, which differentiate them from the nonmotile corynebacteria. Identification of the organism as L. monocytogenes is made by sugar fermentation tests. Treatment Treatment of invasive disease, such as meningitis and sepsis, consists of trimethoprim-sulfamethoxazole. Combinations, such as ampicillin and gentamicin or ampicillin and trimethoprim-sulfamethoxazole, can also be used. Resistant strains are rare. Listeria gastroenteritis typically does not require treatment. LISTERIA MONOCYTOGENES 1 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 13 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ENTEROBACTERIACEAE & RELATED ORGANISMS SALMONELLA Salmonella species cause enterocolitis, enteric fevers such as typhoid fever, and septicemia with metastatic infections such as osteomyelitis. They are one of the most common causes of bacterial enterocolitis Laboratory Diagnosis In enterocolitis, the organism is most easily isolated from a stool sample. However, in the enteric fevers, a blood culture is the procedure most likely to reveal the organism during the first 2 weeks of illness. Bone marrow cultures are often positive. Stool cultures may also be positive, especially in chronic carriers in whom the organism is secreted in the bile into the intestinal tract. Salmonellae form non–lactose-fermenting (colorless) colonies on MacConkey’s or EMB agar. On TSI agar, an alkaline slant and an acid butt, frequently with both gas and H2S (black color in the butt), are produced. S. typhi is the major exception; it does not form gas and produces only a small amount of H2S. If the organism is urease-negative (Proteus organisms, which can produce a similar reaction on TSI agar, are urease-positive), the Salmonella isolate can be identified and grouped by the slide agglutination test into serogroup A, B, C, D, or E based on its O antigen. Definitive serotyping of the O, H, and Vi antigens is performed by special public health laboratories for epidemiologic purposes. SALMONELLA Culture 2 Assist. Prof. Dr. Ameer Mezher Hadi Al-Zahrawi University College Lab. 13 Practical Microbiology Department of Dentisy ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ Microscopic Examination of SALMONELLA SHIGELLA Shigella species cause enterocolitis. Enterocolitis caused by Shigella is often called bacillary dysentery. The term dysentery refers to bloody diarrhea. Laboratory Diagnosis Shigellae form non–lactose-fermenting (colorless) colonies on MacConkey’s or EMB agar. On TSI agar, they cause an alkaline slant and an acid butt, with no gas and no H2S. Confirmation of the organism as Shigella and determination of its group are done by slide agglutination. One important adjunct to laboratory diagnosis is a methylene blue stain of a fecal sample to determine whether neutrophils are present. If they are found, an invasive organism such as Shigella, Salmonella, or Campylobacter is involved rather than a toxin-producing organism such as V. cholerae, E. coli, or Clostridium perfringens. 3 ‫‪Assist. Prof. Dr. Ameer Mezher Hadi‬‬ ‫‪Al-Zahrawi University College‬‬ ‫‪Lab. 13‬‬ ‫‪Practical Microbiology‬‬ ‫‪Department of Dentisy‬‬ ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ ‫‪4‬‬ Al-Zahrawi University College Department of Dentistry Practical Microbiology Lab. 15 Enterobacteriaceae Biochemical Reactions By Assist. Prof. Dr. Ameer Mezher Hadi  IMVIC Test  Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:  The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions  The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae.  IMViC: Indole test § Principle § Certain microorganisms can metabolize tryptophan by tryptophanase § The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia § The presence of indole is detected by addition of Kovac's reagent. Tryptophanase Tryptophane Indole + Pyurvic acid + NH3 amino acids Kovac’s Reagent Red color in upper organic layer`  IMViC: Indole test v Method: Ø Inoculate tryptone water with the tested microorganism Ø Incubate at 37°C for 24 hours Ø After incubation interval, add 1 ml Kovacs reagent, shake the tube gently and read immediately IMViC: Indole test Negative test Positive test e.g. Klebsiella e.g. E. coli v Result: Ø A bright pink color in the top layer indicates the presence of indole Ø The absence of color means that indole was not produced i.e. indole is negative v Special Features: Ø Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-).  IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Neutral pathway Acety methyl carbinol (ACETOIN) Mixed acids  pH less than 4.4 Barrit’s A Methyl Red Barrit;s B indicator MR positive VP positive Pink color Red color E. coli Klebsiella  IMViC test: MRVP test vMethod Ø Inoculate the tested organism into One tube of MRVP broth Ø Incubate the tubes at 37°C for 24 hours Ø AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: Ø Run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3.  For methyl red: Add 6-8 drops of methyl red reagent.  For Voges-Proskauer: Add 12 drops of Barritt's A (- naphthol), mix, 4 drops of Barritt's B (40% KOH), mix  Let sit, undisturbed, for at least 1hour  IMViC test: MR/VP test vResults Methyl Red test Voges-Proskauer test üRed: Positive MR (E. coli) üPink: Positive VP (Klebsiella) üYellow or orange: Negative MR (Klebsiella) üNo pink: Negative VP (E. coli) Principle: Citrate Pyruvate CO2 + Na + H2O Na2CO3 Alkaline,↑pH Simmone’s Citrate media Contains Citrate as a sole of C source Bromothymol blue Positive test Blue colour ØPositive test: Klebsiella, Enterobacter, Citrobacter ØNegative test: E. coli  Citrate Utilization Test Ø Incubate at 37°C for 24 hours.  Citrate Utilization Test Ø Examine for growth (+) Ø Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue Positive Negative Klebsiella, Enterobacter E. coli  Urease Test Ø Urea agar contains urea and phenol red Ø Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 Ø Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. Ø ↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urease H2O Urea CO2 + NH3 NH4 OH ↑ in pH Phenol Red vMethod Pink Positive test Ø Streak a urea agar tube with the organism Ø incubate at 37°C for 24 h  Urease Test vResult  If color of medium turns from yellow to pink indicates positive test.  Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h Positive test Negative test Reaction on Triple Sugar Iron (TSI) Agar  TSI contains  Three different types of sugars ○ Glucose (1 part) ○ Lactose (10 part) ○ Sucrose (10 part)  Phenol red (acidic: Yellow)  TSI dispensed in tubes with equal butt & slant  Principle  To determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide production.  Reaction on TSI  Method:  Inoculate TSI medium with an organism by inoculating needle by stabbing the butt and streaking the slant  Incubate at 37°C for 24 hours  Result Example Reaction on TSI Result Slant Butt H2S color color Non fermenter e.g. Alk/Alk/- Pseudomonas Negative Red Red (No action on sugars) LNF A/Alk/- Negative e.g. Shigella (Glucose fermented Red Yellow without H2S) LNF Positive e.g. Salmonella & A/Alk/+ black in Proteus (Glucose fermented butt Red Yellow with H2S) LF A/A/- e.g. E. coli, Negative (three sugars are Yellow Yellow Klebsiella, fermented) Enterobacter Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae Thank you for listening