19 MIC 206 Differential Tests 2 Inoculation Day S24 PDF

11/5/2024 Differential Tests 2 Inoculations: Extracellular Enzymatic Activities of Microorganisms Urease Test Nitrate Reduction Test Catala...

11/5/2024 Differential Tests 2 Inoculations: Extracellular Enzymatic Activities of Microorganisms Urease Test Nitrate Reduction Test Catalase Test Oxidase Test Utilization of Amino Acids (Phenylalanine) Agglutination Reaction: The Febrile Antibody Test Today’s Inoculation Work as a table Label all tubes, plates, and the test tube rack Do not tighten the caps on inoculated test tubes Labeling Your Tubes Use the white masking tape to label each tube with the following information: – Your table’s symbol – Your section time – The abbreviation of the test – The name of the species the tube is inoculated with – Label your table’s test tube rack. For example, ▪ MW11:50 MRVP E.coli 1  11/5/2024 Biochemistry 2 (PHEN) Phenylalanine slant (NO3 ) Nitrate Broth Escherichia coli Control (Uninoculated) Proteus vulgaris Escherichia coli Orphan Enterococcus faecalis (STR) Starch plate Pseudomonas aeruginosa (Straight line Inoculation) E.coli Escherichia coli Orphan (DNA ) DNA Hydrolysis (DNase plate) Bacillus subtilis (Straight line Inoculation) Orphan Escherichia coli Staphylococcus aureus B. subtilis Your orphan (PHAT) Lipid Hydrolysis (Spirit blue plate) (U) Urea broth Orphan (Straight line Inoculation) Escherichia coli Escherichia coli Proteus vulgaris Bacillus subtilis Orphan Your orphan (GEL) Gelatin Hydrolysis Stab (Blood) Blood Agar Plate Heavy inoculation 2 or 3 loops (Mini Q-streak’s) Escherichia coli Escherichia coli Bacillus subtilis Staphylococcus aureus Your orphan Your orphan Your IA will pick these up for incubation at room temperature. (OX) Oxidase (TSA Plate) (CAT) Catalase (NA Slant) (Mini Q Streak) Enterococcus faecalis Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Orphan Orphan Today’s Inoculation Work as a table Label all tubes, plates, and the test tube rack ▪ MW11:50 MRVP E.coli Do not tighten the caps on inoculated test tubes. Your IA will collect the Gelatin hydrolysis stab for incubation at room temperature. As a super group, combine all the test tubes into one rack. Be sure to label the rack. As a group, tape your 5 plates together, be sure to label the tape. All inoculated material will go into the incubator at 37°C for 16 hours. Identification of Unknown Bacterium Infection? Mix culture, Yes First Step? How does one isolate the causative agent? 2  11/5/2024 Unknown Pathogenic Bacteria Isolated Colonies Mix culture Swab Quadrant Streak Growth Considerations What type of media does one use? Nutrients factors: Vitamins, Minerals, Energy, Carbon Selective, Differential, Complex, or Define? What should be the incubation Temperature? Room, 37˚C, 25˚C, or Body Temperature? What should be the Oxygen conditions for incubation? Aerobic, Anaerobic, or microaerophile environment? Others: pH, Salt…… Unknown Pathogenic Bacteria Isolated Colonies Quadrant Streak Swab A. B. C. Grow each of the colonies as a pure culture. Now, these cultures can be studied to identify the genus and species Next steps? Identification of Physical Characteristics by Staining While we identify each other by physical characteristics, this is not useful with microorganisms. This is due to many organisms being similar. Example: Both E.coli and Salmonella spp. are gram-negative rods. A researcher can’t differentiate them using a microscope. Shape and gram reactions allow a researcher to place the unknown in A. a group. Simple Stain Capsule stain Acid Fast Stain Shape Capsule Mycolic acid Rod Gram Stain Cocci Peptidoglycan Endospore Stain Curved Gram + Spore Former Gram - 3  11/5/2024 Identification Biochemical Characteristics Does the organism possess genes to produces these enzymes or metabolize these molecules? Degradation Enzymes – Catalase Nitrate reductase – Urease Tryptophanase – Lipase Phenylalanine deaminase Sugar Fermentation – Mannitol – Lactose – Glucose Citrate as sole Carbon source Malonate as sole Carbon source Type of Fermentation end products – Methyl red (MR) – Vogus Proskauer (VP) Results of Biochemical RXN Name of the Biochemical Results Results of the Biochemical Tests Test/ Media Do positive results mean more than negative? Catalase POS – NO, they are equal Urea/ Urea broth NEG Phenol red Lactose POS What tests are important? Phenol red Mannitol POS – It depends on how the resource is organized. Simmons Citrate POS Methyl red (MR)/ MRVP broth POS In health care settings, they use a miniaturized test system (see Vogus Proskauer (VP)/MRVP broth NEG below). Indole production POS – They can inoculate many tests simultaneously, incubate the /1% tryptone broth test system, and read the media just like you did for induvial Phenol red Glucose POS test tubes. These cost $100’s per system. Gram Stain NEG KIA/ KIA media POS FTM tube/ fluid thioglycatlate media FAC 0.4% Nutrient Agar MOTILE Starch Hydrolysis/ Starch agar NEG Gelatin Hydrolysis/ Gelatin media NEG Oxidase NEG Nitrate reduction/ Nitrate broth POS Shape POS Type of Hemolysis/ Blood agar NEG Fat hydrolysis/ Spirit blue agar NEG Casein Hydrolysis/ Milk agar NEG H2S production/ KIA media NEG How to use the Bergey’s Manual It would be best if you used the Determinative edition and NOT the Systematic edition. The information commons (lower level of UCENT) has this resource on reserve for two hours at a time. Before checking out the book, you need to fill out the data tables concerning positive or negative reactions. Look at the table of contents to determine what tests should be used at the start of your flow chart. – Gram stain, shape, motility, and oxygen usage. – Note the chapter or group titles. Once you get into a group, you can use the information to get into a genus and determine a species. – Notice that all the Bacillus species are B. megaterium, B. subtilis and B. anthracis are gram- positive, spore formers, and rods. After you have gotten into a group, you choose the tests you want to use to separate the next set of organisms 4  11/5/2024 Bergey’s Homework Assignment Assignment can be found on the canvas site, be sure to download the correct assignment based on your last name. For Bergey’s homework, you should be submitting four items. ✓ Unknown data table (completed) ✓ Unknown flow chart on one page. (Handwritten will not be accepted) ✓ Orphan data table (This is the sheet you have been filling out all semester, handwritten is ok if legible) ✓ Orphan flow chart on one page. (Handwritten will not be accepted) Your flow charts need to identify and separate the unknowns or orphans from the other 26 organisms. Not every single organism from each other. – Before you acquire Bergey’s Manual, which is located on reserve in the information commons (lower level of UCENT), you must complete the data tables. There is no online version. – You need to use the “Determinative” edition of the Manual, NOT the systematic version. – If you are fumbling through the book for 45 minutes and are lost to using the reference book, please see an IA during office hours. – It would help if you were building your flow chart while you are using the Manual. – The flow chart should show a minimum of 7 tests used. – Flow charts that are multiple pages or are cut off will receive point deductions – Use PowerPoint to build your flow chart! It is easier to position lines and boxes. – Notice that all the Bacillus species are B. megaterium, B. subtilis and B. anthracis are gram-positive, spore formers, and rods. – After you have gotten into a group, you choose the tests you want to use to separate the next set of organisms 5