MICROBIAL SYSTEMATICS MBIO 3470 LAB MANUAL 2014 Lab manual is available as a pdf file on the lab website.
MBIO 3470 MICROBIAL SYSTEMATICS SCHEDULE DATE 2014 WEEK EXPERIMENT/PROCEDURE Jan 15 1 Lab Introduction and Standard Operations Procedure (SOP) and BIOSAFETY QUIZ (15 min) - must pass to continue lab. [Not responsible for linked information in lab manual.] Streak plate technique accessed.
MBIO 3470 TABLE OF CONTENTS Lab # Title/Description Page ______________________________________________________________________________ SCHEDULE 2 GENERAL INSTRUCTIONS 4 LAB STANDARD OPERATIONS PROCEDURE 9 PUBLIC HEALTH AGENCY OF CANADA LABORATORY BIOSAFETY GUIDELINES 12 LABORATORY BIOSAFETY GUIDE 13 WHMIS 1 Introduction and Enterobacteriaceae: Biochemical Identification Biosafety Cabinet Operation Part I: Bacteria culture preparation Part II: Enterobacteriaceae: Biochemical Identification Unknown rep
GENERAL INSTRUCTIONS Lab Instructor:Dr. L. Cameron Office: 414B Buller TAs: Scheduled lab time: Jennifer Tanner Other times: Aneil Moya Torres, Elizabeth Hughes, Fraser Ferens Lab Location: 302 Buller Bldg. WEBSITE: http://umanitoba.
4. 5. 6. 7. 8. 9. assignments, and quizzes not collected by the student are destroyed six months after end of term via confidential shredding. Individual lab report total mark may vary (stated on lab report) depending on the complexity and length of the report. All report and quiz values are totalled and brought to maximum 10%. Lab report marks are final unless an obvious error in addition of marks has been made.
5. Must use lab report formats (Word and or Excel) available on lab website. Lab report information is to be presented exactly as requested for each lab report. No binders. Stapled left hand corner. 6. The lab report is to be done as an individual effort. Each student is required to hand in lab reports. Marks will be subtracted for duplication in reports. 7. Cite reference in text of lab report and record full reference at end of lab report.
9. Table presentation (if table format is not available on website) • Table number and title (legend) presented above the table body. • Number tables using Arabic numbers, even if only one table in a report. • Include enough information in title to completely describe table, eliminating the necessity to search elsewhere in the lab report to understand information presented in table. Table title starts with an incomplete sentence.
• • • • • whereas Shigella boydii is abbreviated as S. boydii. If the bacterium’s name also contains a subspecies both the genus and the species may be abbreviated, Aermonas hydrophila anaerogenes as A. h. anaerogenes (this is not standard nomenclature). E. coli is an exception to this rule. Since Escherichia coli has been extensively used in research, it is simply referred to as E. coli for the complete scientific name. The bacterium’s name should not be preceded by an article.
LAB STANDARD OPERATIONS PROCEDURE (SOP) -in compliance with handling of Level II microorganisms. Warning: Many of bacteria used in the MBIO 3470 Systematic’s lab are opportunistic pathogenic bacteria (level II biohazard). This means an immune compromised student may be at risk. Take extreme care following exactly the standard operations procedures. Familiarize yourself with all symptoms in biological MSDS information available in MSDS binder in the lab and online at http://www.phac-aspc.gc.
Lab environment: •Signage (WHIP, Workplace Hazardous Information Plaque) on lab entrance stating biohazard level, lab clothing that must be worn, contact person, entrance requirements (MBIO 3470 students and teaching staff only), Biohazard level, etc. •Posted valid biosafety permit. • Hard copy of all biological MSDS used in the lab. •Lab door must be closed at all times when lab is being used. •A student cannot be in the lab unless supervised by a teaching assistant. Lab is locked at all other times.
•Petri plate containers: All cultures agar plates must be autoclave. Discard culture plates in large plastic lined Petri plate containers. Discard non-sharp biologically contaminated items in the Petri plate container; culture Petri plates, bacterial contaminated paper towels and disposable gloves. •Bench Plastic lined bucket 4: Plastic waste container lined with a clear plastic bag located on your bench or in the biological safety cabinet. All items except Petri Plates and disposable gloves.
PUBLIC HEALTH AGENCY OF CANADA LABORATORY BIOSAFETY GUIDELINES Required Operational Practices for Laboratories handling of infectious materials. 1. Standard Operation Procedures Manual, procedures followed, continually updated. 2. Personnel must receive training. 3. No eating, drinking or smoking. No storage of personal effects in lab. No wearing of jewellery. 4. No oral pipetting permitted. 5. Long hair must be tied back. 6. Lab entry is restricted to authorized personnel. 7.
6. Emergency procedures for spill clean-up, BSC failure, fire, animal escape and other emergencies must be written, easily accessible and followed. A record of people entering the lab during an emergency must be maintained. LABORATORY BIOSAFETY GUIDE UM EHSO Biosafety Guide: http://www.umanitoba.ca/admin/human_resources/ehso/media/BiosafetyGuideMarch05.pdf Both level 1 risk microorganisms and level 2 risk microorganisms are used in this lab.
WHMIS The Workplace Hazardous Materials Information System (WHMIS) is a system for safe management of hazardous materials. WHMIS is legislated by both the federal and provincial governments. Review WHMIS workshop information specific to the Microbiology Department online at http://www.umanitoba.ca/faculties/science/microbiology/WHMISworkshop.htm. Undergraduate students, even though considered workers, do not need to take the quiz.
LAB 1 INTRODUCTION AND ENTEROBACTERIACEAE: Biochemical Identification CLASS II, TYPE A2 BIOSAFETY CABINET OPERATION Labconco Purifier® CELL Logic Room 302-304 supplied with 7 two person Purifier cell logic biological safety cabinets. Introduction The biosafety cabinet is designed to protect you and the environment from exposure to biohazards and to protect samples from contamination during experiment (Figure A). A biosafety cabinet ensures that all biohazard aerosols do not escape into the environment.
Information Centre Panel and Logic Touchpad1 Inside the work area is a LCD information centre panel (figure 1) – frequently check to ensure operating. The Upper Status area should say OK.
fluorescent light turn on. If manual mode, turn on blower and fluorescent light. Allow cabinet to run 3-5 min before using or until ICD information centre panel must show OK. Student Operation of Biosafety Cabinet (BSC). MBIO3470 BSC procedures: All procedure that involves direct handling of MBIO 3470 cultures must be done in BSC (aerosol generating): removal of broth culture cap, inoculation of ID tests, addition of chemicals to ID tests, streaking agar plates, put bacteria on a slide ,etc. 1.
OUTLINE •Lab introduction talk. •15 min BIOSAFETY quiz - Must pass before continuing the lab. Quiz format is mainly short answer, true/false and select correct answer. If a student fails, the next week they will be asked to leave the lab until they can pass a subsequent comprehensive oral test. Mark does not change. The second test is only for admittance to the lab. There will be no opportunity to catch up on work missed.
last name (no initials), plate number (1 or 2), plate type, date, bacteria culture name, “Mixed Culture”. Also streak plates will be assessed for aseptic technique. ENTEROBACTERIACEAE: Biochemical Identification Part I: Bacteria culture preparation Day 1 1. Choose unknown bacterium. RECORD IDENTIFICATION CODE. Sign unknown sheet. Remember to put unknown number or identification code on lab report. 2. Streak two T-soy plates to check for purity and isolation of a single colony (do in BSC).
(iv) DO NOT inoculate biochemical tests from original culture. (v) If you are quite certain that you have identified your unknown bacterium, but one or two tests do not agree. This is expected as some tests just do not give expected results for a particular bacterial species strain but overall results will correctly identify the bacterium. Part II: Enterobacteriaceae: Biochemical Identification The family Enterobacteriaceae5 are gram negative facultatively anaerobic rods.
Report Table 1 for Data Collection. Table 1. Biochemical identification of an unknown Enterobacteriaceae.
LAB 1 REPORT Must use report formats (Word and Excel) available on lab website. Open and save before typing requested information. -Read basic lab report instructions under general instructions. -Read bacteria nomenclature information in general instructions of lab manual. -Remember to underline all bacterial names or if typed use italics. -INDICATE BACTERIUM UNKNOWN CODE on front cover. Data Presentation and Analysis 5 5 1. Record all requested information in lab 1 Table 1.
Table1 . Biochemical Characteristics of selected members of Enterobacteriaceae.
Table2 . Lab 1 Enterobacteriaceae: Biochemical Identification. Report Data Sheet -Record numerical values in the appropriate cells. Record total numerical value. Record bacteria choices at bottom of sheet.
LAB 2 ENTEROBACTERIACEAE: Selective and Differential Media In a clinical lab, seldom is a specimen a pure culture. Selective differential media have been developed to isolate and differentiate members of the family, Enterobacteriaceae. Selective media is defined as media that preferentially isolates a particular group of bacteria. This is usually accomplished by including inhibitors or suppressors of other bacterial groups in the media. Solid agar plates are used in order to isolate pure colonies.
Notes: (i) If you are having problems, it is advisable to streak second plates directly from single colony instead of growing up in broth first. (ii) Often the colour or sheen produced by the bacteria disappears (breaks down) with time (incubating too long or storage in cold box). Read results of plates when requested. 5. Using the T-soy broth culture of each type (pure colony) streak each plate type as illustrated below. Use only one plate of each media type; MacConkey, EMB, Endo, SS, and HE.
LAB 2 REPORT Must use report format (Word) available on lab website. Open and save before typing the requested information.. Lab 2 Enterobacteriaceae: Selective and Differential Media Date: Student Name: Unknown Identification: Table 1. The use of selective and differential media to tentative identify bacteria to the genus level in a mixed Enterobacteriaceae culture. Incubation temperature: Incubation time: Enterobacteriaceae Selective Colony color.
Table 1a: Selective Differential Media for Tentative Identification of Enterobacteriaceae Selection of Enterobacteriaceae and related enteric gram-negative bacteria. Medium and Components Function of Selective and Differential Ingredients Colony Color Analysis MacConkey agar peptone poly peptone lactose bile salts sodium chloride agar neutral red crystal violet distilled water pH 7.1 Bile salts and crystal violet inhibit the growth of gram positive bacteria and fastidious gram negative bacteria.
Table 1b: Selective Differential Media for Tentative Identification of Enterobacteriaceae Selection of Enterobacteriaceae and related enteric gram-negative bacteria. Medium and Components Function of Selective and Differential Ingredients Reaction Interpretation Salmonella-Shigella (SS) agar beef extract peptone lactose bile salts (high) sodium citrate sodium thiosulfate ferric citrate agar neutral red brillant green distilled water pH 7.
LAB 3 ENTEROBACTERIACEAE: API 20E Rapid Identification The methods that you have been using so far to identify bacteria are very time consuming, and therefore not appropriate when results are needed quickly (clinical analysis). Manufacturers have designed a number of simple "test strips" consisting of standard differential identification tests, for example, API-20E, Micro-ID, and Pathotec. The identification systems generate a number based upon the results (-/+).
Identification % id* T* Excellent $99.9 $0.75 Very good $99.0 $0.5 Good $90.0 $0.25 Acceptable $80 $0 Doubtful %id and T given, but a taxon with several tests against the identification is present among those selected (significant taxa). Unacceptable Not given, not close to any taxa in database The identification has low discrimination if 2, 3 or 4 taxa belonging to different genera have been selected.
identification ID and name on the elongated end flap of the bottom incubation tray. Place the API strip in the incubation tray. 4. Inoculation of API Strip: The API strip contains 20 microtubules, each of which consists of a tube and a cupule section. Refer to illustration. Use a sterile Pasteur pipette to add the bacterial suspension, tilt the API incubation tray and place the Pasteur pipette tip against the side of the cupule (load carefully to prevent bubble formation).
enter you best guess and check your results for a valid identification. Remember for a positive VP must be darker than Color Check screen shows for a positive result. Data Collection api 20 Sheet (more available linked on lab website) – not for lab report. 9. After reading discard API strip in Petri plate container or plastic lined bucket on you work bench. Caution: When using the COMPUTER IN THE LAB FIRST REMOVE GLOVES AND WASH HANDS. The TA will monitor you. 10. .
11. Opposite is an example of an apiweb™ identification result printout as requested in lab report. 12. Below is an examples of api 20 E data sheet filled out as requested in lab report.
Table 2a. Chemical and Physical Principles of the API 20E Identification Tests for 18 to 24 hours (apiweb has a color check for negative and positive results on their lab website) TUBE CHEMICAL/PHYSICAL PRINCIPLES [Reactive ingredient is underlined] RESULTS Positive Negative ONPG Hydrolysis of ONPG (colorless) by betagalactosidase releases orthonitrophenol (yellow). IPTG (isopropylthiogalactopyranoside) induces the lac operon.
Table 2b. Chemical and Physical Principles of the API 20E Identification Tests for 18 to 24 hours TUBE CHEMICAL/PHYSICAL PRINCIPLES [Reactive ingredient is underlined] RESULTS Metabolism of tryptophan results in the formation of indole. Kovac’s reagent forms a colored complex (pink to red) with indole. Add 1drop SpotTest Indole Reagent immediate Acetoin, an intermediary glucose metabolite, is produced from sodium pyruvate and indicated by the formation of a colored complex.
LAB 3 REPORT Must use report format (Word) available on lab website. Date: Student Name: Unknown Identification (number or code): Bacterium: 4 1. Record requested identification tests results in the following table. Table 1. API 20 E Identification Test Results for Enterobacteriaceace.
b) Record requested information in the following table. Table 2. Bacterium additional identification test results. Characteristic Test result (record as + or – except for shape) cellular morphology (shape) Gram stain Catalase Motility 2 __ 10 3. Attach apiweb™ identification result printout. Must use printout, not screen printed, to include record of positive and negative results.
LAB 4 ENTEROBACTERIACEAE: Isolation, Identification and Antibiotic Testing The objective of this lab is to use the techniques learned in this lab to identify an Enterobacteriaceae from the environment and most likely demonstrate that this is a difficult task. API manual test system is used to identify the bacterium and antimicrobial susceptibility will be determined using (ATB G-5) strip which contains 21 antibiotics. The first and last pair of cupules are controls.
plate and inoculate a T-soy broth. If you do not have any appropriate isolated colonies, pick from an area that has a possible Enteric and streak for single colonies. Incubate at 37oC overnight. (If necessary, re-streak on T-soy to ensure a pure culture.) Store plates at 4oC (both MacConkey and T-soy plates marked week 7) - do not discard as required for marking. Hints: (i) When selecting a possible Enteric, use information learned so far in this lab.
(available as a Word document on the lab website) Lab 4: Enterobacteriaceae Isolation Data Sheet This table must be completed before scheduled lab for API 20E and antibiotic strip inoculation. See lab schedule.
Week 7 Before proceeding your bacterium must pass all screening tests for Enterobacteriaceae, especially the oxidative-fermentative test. It must be fermentative. 1. Day before lab: Streak plate your tentative Enterobacteriaceae on T-soy agar. Incubate at 37oC overnight. 2. Original MacConkey plates, T-soy streak plate(s) of Enterobacteriaceae and isolation data sheet checked in lab (marks subtracted from lab report if not checked).
ATB G(-) 5 Abbreviations Explanation a Abbreviation Antibiotic/Antibiotic mixture Antibiotic group or Actiona AMO amoxicillin AMC amoxicillin-clavulanic.acidb semi-synthetic penicillins (inhibit bacteria cell wall synthesis) PIC piperacillin TZP piperacillin + tazobactamc TIC ticarcillin TCC ticarcillin-clavulanic.
LAB 4 REPORT Must use report format (Word) available on lab website. Open and save before entering the requested information. Report must be typed unless otherwise stated. Date: Student Name: Isolated Bacterium Name: Bacterium source: 3 1. Record requested information in the following table. Completed table fit on one page. Table 1. Enterobacteriaceae environmental isolation information.
3 2. Attach a copy of apiweb™ identification result printout (includes api 20E cupule data sheet). 2.5 3. a) Completely label and fill out the following ATB G- Antibiotic sensitivity test result sheet. Record bacteria name and source at the top. Include necessary footnotes. Sheet completed in pen acceptable. See procedure and procedure notes to help you completely label and fill out sheet.
1 b) Analyse your ATB G-antibiotic results (select any two antibiotics where your bacterium demonstrates resistance) as requested in the following table. If cephalosporin, indicate generation. # Susceptibility test results 1 Resistance Antibiotic nameb Mode of action of antibiotic. 2a Resistance a If your bacterium shows only one antibiotic resistance just state #2 is not applicable. b include chemical if present 0.5 c) State whether your isolated bacterium would be a clinical threat.
LAB 5 Gram Positive Cocci: MICROCOCCACEAE and STREPTOCOCCACEAE Gram positive cocci (group 17)8 is a diverse group of bacteria. All are gram positive cocci and the majority are non-motile. There are three groups based on oxygen tension; strict aerobes, facultative anaerobes and strict anaerobe. Cell arrangement and the presence of catalase are two main features used to separate genera. In this lab, we will study selected species from the genera Staphylococcus and Streptococcus.
Staphytect Plus Dry Spot Identification Test The diagnostic test, Staphytect Plus, differentiates Staphylococcus aureus strains, which contains coagulase, Protein A and certain capsular polysaccharides, from other staphylococci. These three characteristics of Staphylococcus aureus are assayed to ensure an accurate diagnosis. Staphytect Plus uses blue latex particles coated with porcine fibrogen and rabbit IgG including specific polyclonal antibodies raised against capsular polysaccharides of S. aureus.
Staphytect Plus diagnostic test procedure a) Using a Pasteur pipette add 2 -3 drops of sterile saline to the small rings at the bottom of oval for both the test (your bacteria) and the control. Make sure the saline does not yet mix with the dried latex reagent. It is important that both the control and test be done at the same time to allow comparison. b) CONTROL Move and mix the control saline into the dry Control Latex spots until completely suspended and spread to cover the reaction area.
LAB 5 REPORT Must use report format (Word) available on lab website. Open and save before typing the requested information. Report must be typed and fitted to one page. Date: Student Name: 5 1. Record unknown Staplylococcus or Streptococcus identification data in the following table. Table 1. Test results for the identification of an unknown Staplylococcus or Streptococcus to the species level.
LAB 6 PSEUDOMONADACEAE The family Pseudomonadaceae are gram negative aerobic rods and cocci. All members are motile. Metabolism is respiratory, never fermentative. Strict aerobes. Catalase positive, and oxidase usually positive. For this lab all unknowns are from the genus Pseudomonas that can be identified using API 20 NE strips, most of clinical significance. API 20 NE strip is used to identify non-fastidious, non-enteric gram negative rods such as Pseudomonas.
You need two bacterial suspensions to inoculate the strip, saline and API AUX medium. (i) First prepare a saline suspension of your bacteria suspending ~5 colonies in 2 ml saline. Inoculate tests NO3 to PNG inclusive with saline suspension in tube only, not the cupule. Tilt the strip when adding suspension to tube to help prevent bubble formation. (ii) The API AUX medium supplied by the BioMerieux Company (only one/strip) comes in a glass ampule, open with care. There is a plastic cap.
Table 3. API 20 NE result interpretation. TUBE Active Ingredient/Reaction RESULTS Negative NO3 reduction of potassium nitrate (NO3) to nitrite (NO2) Positive Reagent A and B/within5 min colorless reduction of potassium nitrate (NO3) all the way to nitrogen gas (N2) pink-red Zn/within 5 min pink TRP Metabolism of tryptophan results in the formation of indole.
Table listing possible organisms that can be identified using API 20 NE strip. For each test the expected percentage of positive reactions are given. Greater than 75% is + and less than 25% is negative. (API 20 NE manual.
Sample DATA:
LAB 6 REPORT Must use report format (Word) available on lab website. Open and save before entering the requested information. Report must be typed unless otherwise stated. Student Name: Unknown Identification Code: Unknown Bacterium Name: 4 1. a) Record requested information in the following table. Table 1. Preliminary Screening Test Results for unknown Pseudomonas. Cellular shape Gram stain* Catalase* Motility* * record as + or b) Complete API 20 NE data sheet below.
3 2. Record all requested information in the following table. Table 2. API 20 NE Biochemical Test Results for unknown Pseudomonas. COLOR* RESULT (Incubation time: _____________ ) TUBE NO3 NO2 N2 TRP GLU ADH URE ESC GEL PNG OX** *state exact color, no other details are required except for GEL, record as clear or black without or with diffusion. ** Cytochrome oxidase test (see appendix for protocol) 3 __ 10 3. Include a copy of apiweb identification result printout for 24 h and 48 h (if applicable).
APPENDIX PHASE CONTRAST LIGHT MICROSCOPE OPERATION for ECLIPSE E10010 with SLIDE PHASE CONDENSER Light waves go through viable bacteria almost unchanged. As a consequence unstained bacteria are very difficult to observe using a bright field light microscope. The phase contrast microscope has a phase plate in the objective lens (Ph3 DL x100 1.25 oil immersion) and a matching Ph 4 or Ph3 condenser making viable cells visible by both retarding light waves and reducing amplitude of waves.
9. Adjust the diopters, located at base of each eyepiece to suit your eyes. Holding the top of each eye piece turn clockwise until it stops (standard setting) -lowest setting of each eye piece. First focus on specimen in the usual manner using both eyes and focus knobs. Next focus one eye at a time with respective eyepiece diopter not the focus knobs (turning top of eyepiece counter clockwise, ie., raises eye piece).
work, leave the microscope on top of one of the side benches or the center bench with a note stating problem. TIPS for x1000 Ph4 phase contrast setting: • if you do not use enough oil, a poor microscopic view will occur • fully rack up the condenser for viewing viable cells • set the diopters to match your eyes • light turned up to maximum for viewing viable cells • use a pen marking to focus on Trouble Shooting: No light – check that that cord is fitted tight to the microscope.
BASIC DIFFERENTIAL IDENTIFICATION TESTS Note: If you require a review of basic microbiology procedures please refer to your second year Microbiology lab manual. CELLULAR MORPHOLOGY Microscopically observe and record size (relative only - small, medium, large), shape and arrangement of a fresh sample culture. Refer to the Systematics MBIO 3470 Reference File for details on phase-contrast microscope, wet mount preparation, etc.
GRAM STAIN Introduction: One of the most important and widely used procedures for differentially characterizing bacteria is the gram stain. Bacteria are divided into two groups, based on whether they retain or lose the 'primary stain' (crystal violet) after mordanting with iodine, treatment with alcohol and counter staining with safranin. Gram positive coccoid artifacts may be present in your stained sample. This is an artifact present in the safranin strain, not a contaminant in your bacteria sample.
MOTILITY Bacterial motility may vary with temperature. A. Semisolid agar Introduction: For bacteria that grow rapidly (eg. Enterobacteriaceae), deep tubes containing semisolid agar (0.4%) are most commonly used to check for motility. Tetrazolium salt is added to aid visualization of bacteria motility. The tetrazolium salts (colorless) is converted to an insoluble red formazan by the reducing bacteria.
GENERAL MEDIA T-soy (tryptic soy) agar: 15 g. Bacto tryptone, 5 g. Bacto soytone, 5 g NaCl, 15 g. Bacto agar in 1l iter distilled water. pH6.8 ATB medium: Mueller Hinton broth (300 g. infusion of beef, 17.5 g acidaseTM peptone, 1.5 g starch), 0.05 g CaCl2*, 0.02 g MgSO4*, 1.5 g agar in a final volume 1 liter distilled water. * added separately T-soy medium is a complex medium that allows growth of wide range bacteria as rich in basic requirements, energy, carbon, nitrogen, trace minerals, and vitamins.
BIOCHEMICAL DIFFERENTIAL IDENTIFICATION TESTS Many of the following tests are excerpted directly from the textbook, Color Atlas and Textbook of Diagnostic Microbiology, 2nd edition (1983) by Koneman EW, Allen, SD, Dowell, Jr Vr, and HM Sommers, JB Lippincott company, New York. 1. The majority of the biochemical tests are performed at optimum growth temperature of the bacterium. 2. The incubation time stated in tests are for rapidly growing bacteria.
CATALASE Introduction: The catalase test is used to screen bacteria suspected of belonging to the family Enterobacteriaceae, as all members of this family are positive. However, most aerobic and facultatively anaerobic bacteria (not streptococci) possess catalase activity. Principle: Chemically, catalase is a hemoprotein, similar in structure to hemoglobin, except that the four iron atoms in the molecule are in the oxidized (Fe4+) rather than the reduced (Fe2+) state.
Interpretation: A positive test is represented by the development of a deep blue color anywhere along the slant within 48 hours, indicating that the test organism has been able to utilize the citrate contained in the medium, with production of alkaline products. A positive test may also be read without a blue color if there is visible colony growth along the inoculation streak line.
5. Return the vial to the cupboard. If the vial is not empty, stand the vial upright in the cupboard. If the vial is empty discard in container provided in the cupboard. Interpretation: Positive reaction: immediate appearance (within the first 20 seconds) of purple to black around edge of drop. Negative reaction: bacterium produces no color change or only a change to light grey within 20 second time period. Disregard all color changes after 20 seconds.
Media and reagents: Decarboxylase medium base (Falkow used for Enterobacteriaceae and Pseudomonas) peptone 5g yeast extract 3g dextrose 1g bromocresol purple 0.0 2 g distilled water to 1 liter Final pH = 6.8 Amino acid: 0.5% L form of amino acid. Procedure: Inoculate the test amino acid and control decarboxylase tube with test organism (through the mineral oil layer into medium). Prior to use, the broth tubes are stored at 4oC. Incubate at optimum temperature for 2 days.
GELATIN LIQUEFACTION Introduction: Gelatin is a complex derivative of animal collagen that has poor nutritive value and is used in culture media to test the ability of organisms to produce gelatinase. Principle: Gelatinases are proteolytic enzymes capable of hydrolysing gelatin so that its ability to form a gel is lost.
HYDROGEN SULFIDE PRODUCTION Introduction: The ability of certain bacteria to liberate sulfur in the form of H2S from sulfur containing compounds is an important characteristic for identification. Principle: H2S can be detected in a test system if the medium contains a source of sulfur (sodium thiosulfate, peptone containing cysteine and methionine), an H2S indicator (ferrous sulfate), the indicator phenol red and basal medium that supports the growth of the bacteria.
INDOLE Introduction: Indole, a benzyl pyrrole, is one of the metabolic degradation products of the amino acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolysing and deaminating tryptophan with the production of indole, pyruvic acid, and ammonia.
METHYL RED (MR) Introduction: Methyl red is a pH indicator with a range between 6 (yellow) and 4.4 (red). The pH at which methyl red detects acid is considerably lower than the pH for other indicators used in bacteriologic culture media. Thus, in order to produce a color change, the test organism must produce large quantities of acid from the carbohydrate substrate being used.
NITRATE REDUCTION Introduction: The capability of an organism to reduce nitrate to nitrites is an important characteristic used in the identification and species differentiation of many groups of microorganisms. All Enterobacteriaceae except certain biotypes of Enterobacter agglomerans and Erwinia demonstrate nitrate reduction. Principle: Organisms demonstrating nitrate reaction have the capability of extracting oxygen from nitrates to form nitrites and other reduced products.
OXIDATIVE-FERMENTATIVE TEST (Hugh and Leifson) Introduction: Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively. The end products of fermentation are relatively strong mixed acid that can be detected in a conventional fermentation test medium. However, the acids formed in oxidative degradation of glucose are extremely weak, and the more sensitive oxidative-fermentation medium of Hugh and Leifson (OF medium) is required for detection.
PHENYLALANINE DEAMINASE Introduction: Phenylalanine is an amino acid that upon deamination forms a keto acid, phenylpyruvic acid. Of the Enterobacteriacaeae, only members of Proteus possess the deaminase enzyme necessary for this conversion. Principle: The phenylalanine test depends upon the detection of phenylpyruvic acid in the test medium after growth of the test organism. The test is positive if a visible green color develops upon addition of a solution of 10% ferric chloride.
VOGES-PROSKAUER TEST Introduction: Two microbiologists working at the beginning of the 20th century first observed the red color reaction produced by appropriate culture -media after treatment with potassium hydroxide. It was later discovered that the active product in the medium formed by bacterial metabolism is acetyl-methyl carbinol, a product of the butylene glycol pathway.
FINAL LAB EXAM: Microbiology MBIO 3470 DATE: Sample lab exam PAGE: 1 of 3 INSTRUCTOR: Dr. L. Cameron STUDENT NAME: ____________________ MICROBIAL SYSTEMATICS TIME: 2 h STUDENT NUMBER: ________________ WRITE EXAM IN PEN ONLY. BRIEFLY ANSWER ALL QUESTIONS IN SPACE PROVIDED ONLY POINT FORM IS ACCEPTABLE. Spaces of example exam have been compressed to shorten exam for reference file. All necessary IDENTIFICATION TABLE SUPPLIED. RETURN TABLES WITH EXAM.
FINAL LAB EXAM: Microbiology MBIO 3470 DATE: April 3, 1996 2 of 3 INSTRUCTOR: Dr. L. Cameron STUDENT NAME: ______________________ MICROBIAL SYSTEMATICS TIME: 2 h STUDENT NUMBER: __________________ 3 3. (a) The general procedure for unknown bacteria determination is to inoculate biochemical tests with colonies of unknown bacterium. Name agar medium? Explain why this medium is used.
FINAL LAB EXAM: Microbiology MBIO 3470 MICROBIAL SYSTEMATICS DATE: April 3, 1996 PAGE: 3 of 3 TIME: 2 h INSTRUCTOR: Dr. L. Cameron STUDENT NAME: __________________ STUDENT NUMBER: __________________ 6 6. What identification test (media/chemicals added) may be performed to show the following bacterial characteristic? Include appearance of positive/negative test results.
FINAL LAB EXAM: Microbiology MBIO 3470 MICROBIAL SYSTEMATICS DATE: April 3, 1996 PAGE: 4 of 4 TIME: 2 h INSTRUCTOR: Dr. L. Cameron STUDENT NAME: ______________________ STUDENT NUMBER: __________________ 1 10. 6 a) API 20 NE often is read at both 24 hours and 48 hours after incubation at 28oC. Explain why. b) Record requested information in the following table API 20 NE Test Test demonstrates (principle) (Include chemical(s) added if applicable) Positive test result PNG ARA PAC 5 11.
