Microlab ch 1.pdf

A Comparison of Ill Hand-Cleansing Agents 1 Theory The concept of good hand hygiene has gone from a been shown to result i...

A Comparison of Ill Hand-Cleansing Agents 1 Theory The concept of good hand hygiene has gone from a been shown to result in a higher level of compliance by controversial beginning (in the early 1800s) to an health-care workers. Examples of agents that can be accepted practice that is still problematic. Current studies tested in this exercise are shown in Figure 1.1. designed to test the efficacy of various agents often have subjects wash for unrealistic lengths of time (that is, longer than workers routinely wash on the job), test Application artificially contaminated hands (or not), and use different Effective hand washing to minimize direct person-to- standards of evaluation, making comparison difficult. person transmission, and indirect contact of pathogens by We are still left with the question, "What works best?" health-care professionals and food handlers, is essential. While it has been identified as an important, easily It also is critical to laboratorians handling pathogens to performed behavior that minimizes transfer of pathogens minimize transmission to others, inoculation of oneself, to others, uniform compliance with hand-washing stan- and contamination of cultures. dards has been difficult to achieve. Heavy workloads, skin reactions to the agent (e.g., plain or antimicrobial soap, iodine compounds, alcohol), skin dryness due to frequent In This Exercise washing, and many other factors contribute to noncom- You will evaluate the relative effectiveness of a variety of pliance (see Boyce and Pittet, 2002, and The Joint hand-washing agents. Commission, 2009). Alcohol-based hand rubs have, in many instances, replaced conventional hand-washing agents because they.I Materials are more effective than soap and water (disputed), Per Student require less time, produce fewer skin reactions, and have D One nutrient agar or tryptic soy agar plate D Access to a sink and paper towels 1 Thanks to reviewers Johana Meledez-Santiago and Janice Yoder Smith for their suggestions on developing this laboratory exercise. D Permanent marking pen 1.1 Hand-Washing Agents + From left to right are: Betadine® scrub (10% povidone iodine), alcohol-based hand sanitizer (65% ethyl alcohol), 0.13% benzalkonium chloride-based hand sanitizer, antibacterial soap (0.15% triclosan), and Hibiclens® (4% chlorhexidine gluconate). As written, this exercise uses these agents, but others can be substituted, including tap water. II] PerBottles Table D of as many as five of the following or suitable a Place a quarter-size drop of the first hand-cleansing agent in the palm of your left hand. Rub your right replacements: thumb on the area to be sampled. Then, rub your Antibacterial liquid soap (0.15% triclosan) thumb into the agent for 10 seconds. 2 (If you don't have a watch, sing the "Happy Birthday Song" Hand sanitizer (65% ethyl alcohol = 65% ethanol) once to yourself.) Once your thumb is dry, gently Hy5 brand of alcohol-free hand sanitizer press it next to the number 1 on the "After" side. (0.13% benzalkonium chloride) Wash both hands with soap and water, and dry Betadine® scrub (10% povidone iodine) them. Hibiclens® (4% w/v chlorhexidine gluconate) b Repeat step a with each finger of the right hand Tap water and the remaining four hand-cleansing agents. Note: Students should not ingest or get any of these Be sure to press each finger onto the agar next products in their eyes. Follow package inserts for treat- to its correct number and to wash your hands in ments. Students with allergies or sensitivities to the ac- between exposures. tive ingredients in any of these products should be 7 Tape the lid on the plate and incubate at 25°C until assigned an alternate product. the next lab period. Lab Two 1 Examine your plates for growth. (Note: don't remove Procedure the plate's lid unless given permission to do so.) Lab One Each "blob" is probably the product of a single mi- 1 Label the base of a nutrient agar or tryptic soy agar crobial species that has reproduced from one or a plate with your name. Divide the plate in half and few cells to such an extent that it is now visible to label one half "Before" and the other half "After." the naked eye. This is called a "colony." Different Then, number 1 through 5 on both sides of the di- microbial species often produce distinctly different viding line so the numbers line up. That is, number 1 colonies. on the "Before" side should be opposite number 1 on 2 Fill in the table provided on the data sheet, page 19, the "After" side, and so on. Use the entire diameter of as you evaluate the relative amount of growth and the plate for the five numbers and spread them out microbial diversity (based on colony differences) on as much as you can. You will be pressing your fingers the "Before" and "After" sides of the plate for each at each number in steps 3 and 6. agent. Recognize that you are comparing "apples 2 Rub your left hand fingertips (all five of them) on and oranges" with respect to their intended uses. the area to be sampled. (Good choices are the floor, That is, the products tested range from surgical shoe soles, tabletop near a sink, backpack, and your scrubs to over-the-counter soaps intended for forehead or feet. Be creative! household use. A surgical scrub may have better 3 Then, one at a time, gently press your thumb tip antimicrobial activity than hand soap, but that next to the "Before" number 1, your index finger doesn't make it "better" for household use! next to the "Before" number 2, and so on. Your 2 middle, ring, and pinky fingers are numbered 3, 4, The recommended time for hand washing is 15-20 seconds, but because you are only washing the fingertips and concentrating on those, that and 5, respectively. time has been reduced in order to see representative results. 4 Wash your hands with soap and water. 5 Record the hand-cleansing agents you will be using References on the data sheet, page 19. Boyce, John M. and Didier Pittet. Centers for Disease Control and 6 Now sample the same site used in step 2 with Prevention. Guideline for Hand Hygiene in Health-Care Settings: the fingertips of your right hand, but this time one Recommendations of the Health care Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand fingertip at a time. Then, you will rub that fingertip Hygiene Task Force. MMWR 2002, 51 (No. RR-16): 1-45. in a few milliliters of the hand-cleansing agent in your The Joint Commission, 2009. Measuring Hand Hygiene Adherence: left palm (see steps a and b for further directions). If Overcoming the Challenges. the agent requires rinsing and drying, perform this http://www.jointcommission.org/assets/1/18/hh_monograph.pdf. at a sink. Agents that only require air-drying can be done at your table.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - A Comparison of Hand-Cleansing Agents OBSERVATIONS AND INTERPRETATIONS 1 Record your results in the table below. For relative amount of growth, use this qualitative scale for evaluation: +++ means "a lot of contamination" ++ means "moderate contamination" + means "little contamination" 0 means "no contamination" There is no absolute cutoff between any of these categories. What you call "moderate contamination" might be called "little contami- nation" by another student. Just try to be consistent within your evaluation. For diversity, look for different colors, shapes, textures, sizes, and anything else that is indicative of a different organism growing, and record the number of different organisms present. Interpretation would be relative effectiveness of the agent (excellent, good, fair, poor). "Before" "After" Hand-washing Agent ---- Interpretation 2 3 4 5 QUESTIONS 1 Which agent seemed to be most effective at removing microbes from the fingertips? SECTION 1 Fundamental Skills for the Microbiology Laboratory 19 -.. _r --1..r ' ,J. -·./ ~. ~·.. ) ,Jr II] 2 Which agent seemed to be least effective at removing microbes from the fingertips? 3 What other variables besides the cleansing agent might be responsible for any differences noted between fingers or students? 4 Did you or any other students notice some microbes persisting more than others after washing? What factor(s) might account for this? 5 Do some research and fill in the following table. Your answer to "Antibacterial Effect" should address the structural and/or functional part of the cell affected, as well as if the agent is bactericidal (kills bacteria) or bacteriostatic (stops bacterial growth, but doesn't kill them). Under "Other Antimicrobial Effects," note any antiviral, antiprotozoal, and/or antifungal properties of the agent. Active Ingredient Antibacterial Effect Other Antimicrobial Effects Triclosan lsopropanol/ethanol (isopropyl/ethyl alcohol) Benzalkonium chloride Povidone iodine scrub Chlorhexidine gluconate... _~ 20 MICROBIOLOGY: Laboratory Theory & Application.._ _,,,.. ' ·' '-. If#.,,  Basic Growth Media Ill To cultivate microbes, microbiologists use a variety of growth media. Although these media may be formulated from scratch, they more typically are produced by rehydrat- ing commercially available powdered media. Media that are routinely encountered in the microbiology laboratory range from the widely used, general-purpose growth me- dia, to the more specific selective and differential media used in identification of microbes (Sections 4 and 5). In Exercise 1-2 you will learn how to prepare simple general growth media. + EXERCISE Nutrient Broth and Nutrient Agar Preparatio_n_ _ _ _ __ 1-2 Theory In This Exercise Nutrient broth and nutrient agar are common media You will prepare 1-liter batches of two general growth used for maintaining bacterial cultures. To be of practical media: nutrient broth and nutrient agar. During the course use, they have to meet the diverse nutrient requirements of the semester, a laboratory technician will probably do of routinely cultivated bacteria. As such, they are formu- this for you, but it is good to gain firsthand appreciation lated from sources that supply carbon and nitrogen in a for the work done behind the scenes! variety of forms-amino acids, purines, pyrimidines, monosaccharides to polysaccharides, and various lipids. Generally, these are provided in digests of plant material./ Materials (phytone) or animal material (peptone and others). Be- Per Student Group cause the exact composition and amounts of carbon and D One 2-liter Erlenmeyer flask for each medium made nitrogen in these ingredients are unknown, these media D Three or four 500 mL Erlenmeyer flasks and covers are considered to be undefined. They are also known as (can be aluminum foil) complex media 1 o Stirring hot plate In most classes (because of limited time), media are D Magnetic stir bars prepared by a laboratory technician. Still, it is instructive for novice microbiologists to at least gain exposure to D All ingredients listed in the following recipes what is involved in media preparation. Your instructor (or commercially prepared dehydrated media) will provide specific instructions on how to execute this D Sterile Petri dishes exercise using the equipment in your laboratory. D Test tubes (16 mm X 150 mm) and caps o Balance D Weighing paper or boats Application D Spatulas Microbiological growth media are prepared to cultivate microbes. These general growth media are used to main- tain bacterial stock cultures. Medium Recipes Nutrient Broth 1 Beef extract 3.0 g In contrast, chemically defined media are composed of ingredients with a known chemical structure and in known quantities. For an example, see Peptone 5.0 g the recipe for Simmon's Citrate Agar on page 864. In it, the only carbon Distilled or deionized water 1.0 L source is the six-carbon sugar citrate (C 6 H 8 0 7 ) and the only nitrogen source is ammonium (NH 4+). pH 6.6-7.0 at 25°C II] Nutrient Agar Beef extract 3.0 g 6 Incubate the slants and/or deep tubes at 35 ± 2 °C for 24 to 48 hours. Peptone 5.0 g Nutrient Agar Plates Agar 15.0 g 1 Weigh the ingredients on a balance (Fig. 1.2). Distilled or deionized water 1.0 L 2 Suspend the ingredients in 1 liter of distilled or pH 6.6-7.0 at 25°C deionized water in the 2-liter flask, mix well, and boil until fully dissolved (Fig. 1.3 ). Preparation of Medium Lab One To minimize contamination while preparing media, clean the work surface, turn off all fans, and close any doors that might allow excessive air movements. Nutrient Agar Tubes 1 Weigh the ingredients on a balance (Fig. 1.2). 2 Suspend the ingredients in 1 liter of distilled or deionized water in the 2-liter flask, mix well, and boil until fully dissolved (Fig. 1.3). 3 Dispense 7 mL portions into test tubes and cap loosely (Fig. 1.4). If your tubes are smaller than those listed in Materials, adjust the volume to fill 20% to 25% of the tube. Fill to approximately 50% for agar deeps. 4 Sterilize the medium by autoclaving for 15 minutes at 121°C (Fig. 1.5). 5 After autoclaving, cool to room temperature with the tubes in an upright position for agar deep tubes. Cool with the tubes on an angle for agar slants (Fig. 1.6). 1.3 Mixing the Medium + The powder is added to a flask of distilled or deionized water on a hot plate. A magnetic stir bar mixes the medium as it is heated to dissolve the powder. 1.2 Weighing Medium Ingredients + Solid ingredients are weighed with an analytical balance. A spatula is used to transfer the powder to a tared weighing boat. Shown here is dehydrated nutrient 1.4 Dispensing the Medium into Tubes +An adjustable agar, but the weighing process is the same for any powdered pump can be used to dispense the appropriate volume (usually ingredient. 7 ml-10 ml) into tubes. Then, loosely cap the tubes.  3 Divide into three or four 500 mL flasks for pouring. Smaller flasks are easier to handle when pouring 3 Dispense 7 mL portions into test tubes (or less, depending on your lab customs) and cap loosely Ill plates. Don't forget to add a magnetic stir bar and (Fig. 1.4). As with agar slants, if your tubes are to cover each flask before autoclaving. smaller than those recommended in Materials, 4 Autoclave for 15 minutes at 121°C to sterilize the add enough broth to fill them approximately 20% medium. to 25%. 5 Remove the sterile agar from the autoclave, and 4 Sterilize the medium by autoclaving for 15 minutes allow it to cool to 50°C while it stirs on a hot plate. at 121 °C (Fig. 1.5). 6 Dispense approximately 20 mL into sterile Petri plates (Fig. 1. 7). Be careful! The flask will still be hot, so wear an oven mitt. While you pour the agar, shield the Petri dish with its lid to reduce the chance of introducing airborne contaminants. If necessary, gently swirl each plate so the agar completely covers the bottom; do not swirl the agar up into the lid. Allow the agar to cool and solidify before moving the plates (Fig. 1.8). 7 Store these plates on a countertop for 24 hours to allow them to dry prior to use. Nutrient Broth 1 Weigh the ingredients on a balance (Fig. 1.2). 2 Suspend the ingredients in 1 liter of distilled or deionized water in the 2-liter flask. Agitate and heat slightly (if necessary) to dissolve them completely (Fig. 1.3). 1.6 Tubed Media + From left to right a broth, an agar slant, and an agar deep tube. The solid media are liquid when they are removed from the autoclave. Agar deeps are allowed to cool and solidify in an upright position, whereas agar slants are cooled and solidified on an angle. 1.5 Autoclaving the Media + Media are sterilized in an autoclave for 15 minutes at 121 °C. Shown are four racks of tubed media being removed from an autoclave. The tubes on the left are brain-heart infusion broth (BHI) and they will be allowed to stand and cool. Once cool, they will remain liquid. The tubes on the right contain brain-heart infusion agar (BHIA), a solid medium, which is liquid when it is re- moved from the autoclave but will solidify as it cools due to the agar in it. The position of the tube as it cools will determine whether an 1.7 Pouring Agar Plates + Agar plates are made by pouring agar deep tube or an agar slant is produced (Fig. 1.6) Note the sterilized medium into sterile Petri dishes. The lid is used as a angled sides of the racks that allow them to be tipped so the agar shield to prevent airborne contamination. Once poured, the dish will solidify in a slant. Plated media are autoclaved in a flask and then is gently swirled so the medium covers the base. Plates are then dispensed into sterile Petri dishes (Fig. 1 7). cooled and dried to eliminate condensation. a J LabTwo Reference 1 Examine the tubes and plates for evidence of growth. Zimbro, Mary Jo and David A. Power. Pages 404-405 and 408 in DIFCQTM & BBLTM Manual-Manual of Microbiological Culture 2 Record your observations on the data sheet, page 25. Media. Sparks, MD: Becton, Dickinson and Company, 2003. 1.8 An Agar Plate + Plated media are often used for isolating individual species from a mixed culture (Exercises 1-4 and 1-5) or for counting the number of cells in a diluted sample (Exercise 6-1 ). Some differential tests also use plated media (e.g., milk agar, DN- ase agar, starch agar). Shown is a BHIA plate, which stands for "brain-heart infusion agar." The 1.5%-2% agar in the medium acts as a solidifying agent to suspend the nutrients-extracts of brain and heart tissues. Brain and heart provide carbon, nitrogen, and other essential nutrients for growth, as well as energy.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Nutrient Broth and Nutrient Agar Preparations OBSERVATIONS AND INTERPRETATIONS 1 In the following table, record the number of each medium type you prepared, and then record the number of apparently sterile ones. Calculate your percentage of successful preparations for each. In the last column, speculate as to probable/possible sources of contamination (even if all your media are sterile). Number Percentage. of Sterile of Successful Medium I Preparations Preparations Probable Sources of Contamination Nutrient agar tubes (slant or deep) Nutrient agar plates Nutrient broths QUESTIONS 1 Which medium was most difficult to prepare without contamination? Why do think this might be so? SECTION 1 Fundamental Skills for the Microbiology Laboratory 25... r ·-1..r ' ,J. -·./ ~. ~·.. ) ,Jr II] 2 For each of the following types of contamination suggest the most likely point in preparation (or later) at which the contaminant was introduced. a. Growth in all broth tubes. b. Growth in one broth tube. c. Growth only on the surface of a plate. d. Growth throughout the agar's thickness on a plate. e. Growth only in the upper 1 cm of agar in an agar deep tube. £. All plates in a batch have the same type and density of contaminants. g. Only a few plates in a batch are contaminated, and each looks different.... _~ 26 MICROBIOLOGY: Laboratory Theory & Application.._ _,,,.. ' ·' '-. If#.,,  Aseptic Transfers and Inoculation Methods Ill As a microbiology student, you will be required to transfer living microbes from one place to another aseptically (i.e., without contamination of the culture, the sterile medium, or the surroundings). While you won't be expected to master all transfer methods right now, you will be expected to perform most of them over the course of the semester. Refer to Exercises 1-3 through 1-5 and Appendices 8, C, and Das needed. To prevent contamination of the sample, inoculating instruments (Fig. 1.9) must be sterilized prior to use. Wire inoculating loops and needles are sterilized immediately before use in an incinerator or Bunsen burner flame. The mouths of tubes or flasks containing cultures or media are also incinerated at the time of transfer by passing their openings through a flame. Instruments that are not conveniently or safely incinerated, such as Pasteur pipettes, cotton applicators, glass pipettes, and digital pipettor tips, are sterilized inside wrappers or containers by autoclaving prior to use. Aseptic transfers are not difficult; however, a little preparation will help assure a safe and successful procedure. Before you begin, you will need to know where the sample is coming from, its destination, and the type of transfer instrument to be used. These exercises provide step-by-step descriptions of routine transfer methods. Certain less-routine transfer methods are discussed in Appendices 8 through D. + 1.9 Inoculating Instruments + Any of several different instruments may be used to transfer a microbial sample, the choice of w hich depends on the sample source, its destination, and any special requirements imposed by the specific protocol. Shown here are several examples of transfer instruments. From left to right: serological pipette (see Appendi x C), disposable transfer pipette, Pasteur pipette, inoculating needle, inoculating loop, disposable inoculating needle/loop, cotton swab (see Appendix Band Exercise 1-4), and glass spreading rod (see Exercise 1-5). (Note: the glass rod is not an inoculation instru- ment, but it is used to spread an inoculum introduced to an agar plate by another instrument. As such, it is an instrument used in an inoculation process.) When transferring BSL-2 organisms, we advise using a sterile disposable loop or wooden stick (not shown). Neither of these requires incineration after use and minimize the threat of aerosol production.  Common Aseptic Transfers EXERCISE Ill and Inoculation Methods 1-3 Theory A medium that contains living microbes is called a culture. Be organized. Arrange all media in advance and clearly If a culture contains a single species it is said to be a pure label them with your name, the date, the medium, and culture. It is essential to transfer microbes from their pure the inoculum (Fig. 1.10). Tubes may be labeled with culture to a sterile medium aseptically, that is, without tape, paper held on with a rubber band, or by writing contamination of yourself, others, the environment, the directly on the glass (this option presumes you will source culture, or the medium being inoculated. In other clean the writing off before you dispose of the culture words, you want your pure culture to stay pure, your for decontamination). You should write directly on the new culture to be pure, and the surroundings to remain base (not the lid) of plastic Petri plates because they are uninocula ted. disposable. Be sure not to place any labels in such a Following are general techniques and practices that way as to obscure or obstruct your view of the tube's improve your chances of successfully making an aseptic or plate's interior. transfer. Equally important are the techniques related to Place all media tubes in a test tube rack when not in use safety. It has been demonstrated that while"... the causative whether they are sterile or not. Tubes should never be incident for most LAls (laboratory acquired infections) is laid on the table surface because they may leak (Fig. unknown... A procedure's potential to release microor- 1.11). ganisms into the air as aerosols and droplets is the most Take your time. Work efficiently, but do not hurry. You important operational risk factor... " (Chosewood and are handling potentially dangerous microbes. Working Wilson, 2009). In other words, limiting aerosol production at a frenzied pace leads to carelessness and accidents. is a safety issue and not an issue of keeping pure cultures pure. Please adhere to these practices for your safety and Never hold a tube culture by its cap. Caps are generally the safety of those around you. loose to allow aeration and are not secure enough to be used as a handle. Even screw caps can be loose Minimize the potential of contamination. Do not enough so as not to be secure. perform any transfers over your books and papers because you may inadvertently and unknowingly Hold the inoculating loop or needle like a pencil in contaminate them with droplets or aerosols that settle. your dominant hand and relax (Fig. 1.12)! Put them safely away. Some labs advise performing Adjust your Bunsen burner so its flame has an inner transfers over a disinfectant-soaked paper towel. and outer cone (Fig. 1.13). 1.10 Label The Media + To avoid confu- sion after-the-fact, it is best to label sterile media prior to inoculating it. (A ) Tubed media can be labeled with tape, paper labels, or directly on the glass with a marking pen. Labels must be removed when tubes are put in the autoclave bin for sterilization. (B) Plastic Petri dishes should be labeled on their base, not on their lid, because the lid may get separated from its base during reading or rotated from its correct orientation. Because most labs use disposable Petri dishes, the labels do not have to be removed prior to autoclaving. II] 1.11 Microbiologist at Work + Materials are neatly positioned and not in the way. To prevent spills, culture tubes are stored upright in a test tube rack. They are never laid on the table. The microbiologist is relaxed and ready for work. Notice he is holding the loop like a pencil, not gripping it like a dagger. 1.13 Bunsen Burner Flame + When properly adjusted, a Bunsen burner produces a flame with two cones. Sterilization of inoculating instruments is done in the hottest part of the flame- the tip of the inner cone (red arrow) Heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items may be done in the outer cone (white arrow) 1.12 Hold the Loop Like a Pencil +Holding the loop as shown puts the hand in a convenient position to hold tube caps by the "pinky" finger pure culture. (A streak plate and sometimes a spread plate are exceptions to this. See Exercises 1-4 and 1-5.) Transfers can be made between all forms of media- Application slants, broths, and plates-depending on the intended use Media come in many forms, each with specific applica- of the new culture. The following is organized into transfers tions. Broths are used to grow microbes when fresh from broth culture to sterile broth, agar slant culture to cultures or large numbers of cells are required. Broths of sterile agar slant, and plate culture to sterile broth. If you differential media are also used in microbial identification can do these, then you have the skills to transfer between (Section 5). Agar slants are generally used to grow stock most any combination of media. (Inoculation of sterile cultures that can be refrigerated after incubation and agar plates is covered in Exercises 1-4 and 1-5 and will maintained for several weeks. In addition, many differ- complete your skill set.) ential media are agar slants. Plated media are typically used The instruments usually used for transfers are either for obtaining isolation of species (Exercises 1-4 and 1-5), inoculating loops or inoculating needles. (Pipettes are differential testing, and quantifying bacterial densities also sometimes used and these are covered in Appendices (Exercise 6-1 ). In all cases, using these media requires C and D.) For simplicity, the following instructions only aseptic inoculation in which a portion of an existing pure refer to inoculating loops, but the same apply to inocu- culture is transferred to a sterile medium to start a new lating needles. A Special Note about Transferring BSL-2 Organisms Wearing gloves and eye protection are also recommended. Ill Most college biology teaching laboratories have eliminated or reduced the use of BSL-2 organisms, as we have in this Identifying BSL-2 organisms with red caps on tube edition of the lab manual. However, use of some BSL-2 cultures or BSL-2 labels on plate cultures. organisms is unavoidable for some tests. In other exercises, Whatever precautions your college's guidelines dictate, they are included as optional test organisms. take them seriously. Your health and the health of others The primary concern with BSL-2 organisms is con- in your lab are at stake. tamination or infection due to inhalation of aerosols. Aerosols are problematic because we generally are unaware Transfer from a Broth Culture to a Sterile Broth of their production and they remain suspended in the air As you read these instructions, also follow the procedural long after the procedure has been completed. Throughout diagram in Figure 1.15 to get a summary view of the this section we emphasize techniques that minimize aerosol process. Make appropriate adjustments if handling a production, but they are even more essential when han- BSL-2 organism. dling BSL-2 organisms. Your lab will have specific guide- 1 Label the sterile broth tube with your name, the lines on how to handle BSL-2 organisms and you should date, and the organism you are inoculating it with. take these seriously. They may include any or all of the following precautions, depending on the exercise and the 2 Make sure your loop is a closed circle. If it isn't, pinch equipment you have available. it closed. Hold it like a pencil, and then flame it from base to tip as shown in Figure 1.16. Be sure the entire Performing tests in a Class II biosafety cabinet (Fig.1.1) wire reaches orange-hot at some point. is recommended, but not all teaching laboratories have these installed. 3 Suspend the bacteria in the broth culture with a Using an electric incinerator (Fig. 1.14) for decontami- vortex mixer prior to transfer (Fig. 1.17). Be sure nating wire loops and needles, which simultaneously not to mix so vigorously that broth gets into the contains aerosols within the ceramic interior and cap or that you lose control of the tube. Start slowly, decontaminates them. and then gently increase the speed until the tip of the vortex reaches the bottom of the tube. Alternatively, Using sterile disposable loops/needles or wooden the culture may be agitated by drumming your fingers sticks that don't require flaming after use and can be along the length of the tube several times (Fig. 1.18 ). disposed of in sharps containers or other appropriate Again, be careful not to splash broth into the cap or receptacles for autoclaving. lose control of the tube. 4 Loosen the cap of the culture tube (this is especially important if you are using a screw-cap tube). Move the culture tube to your loop hand. Remove and hold the tube's cap with the little finger of your loop hand (Fig. 1.19). Moving the tube and not the loop prevents excessive movement of the loop that might result in aerosols or droplets. 5 Hold the tube on an angle to minimize the opportu- nity for airborne contamination (Fig. 1.20). 6 Incinerate the lip of the tube by passing it quickly through the flame two or three times (Fig. 1.21). Do not wave it through the flame so fast that broth sloshes out of the open end. 1.14 Bacteriological Incinerator + Bacterial incinerators use infrared heat and reach temperatures over 800°C. A wire loop/needle is inserted into the incinerator and heated until the wire is red-hot (usually within 5-7 seconds). (The handle may also get hot, so be careful.) The loop/need le is then removed and allowed to cool with- out touching anything. It may then be used to transfer microbes, or if this is done at the completion of a transfer, it may be set aside until needed again. II] 1 Label the sterile broth 2 Hold the loop like a 3 Flame the loop and 4 Pick up the culture tube with your name, pencil in your domi- wire from base to tube in your free hand the date, the medium, and the organism's....... nant hand tip; make sure it gets uniformly hot and suspend the organisms in the broth name t 8 Move the tube over 7 Flame the tube's lip 6 Hold the tube on an 5 Remove the tube's cap the loop until the loop by passing it quickly angle with the little finger of is in the broth through the flame two your loop hand or three times {do not splash the broth out of the open tube) t 9 Remove the tube from 10 Keeping the loop 11 Return the tube to 12 Pick up the sterile over the loop, being hand still, flame the the test tube rack broth tube in your careful not to catch the tube's lip as before free hand loop on the tube's lip and replace the cap (the loop should have a film of broth in it) t 16 Move the tube over 15 Flame the tube's lip 14 Hold the tube on an 13 Keeping the loop the loop until the by passing it quickly angle hand still, remove the loop is in the broth through the flame +-, tube's cap with the +-i +-i two or three times little finger of your loop hand t 17 Mix the inoculum in 18 Withdraw the tube so 19 Keeping the loop 20 Return the tube to the sterile medium the loop is out of the hand still, flame the the test tube rack....... broth, then remove the film in the loop tube's lip as before and replace the cap by tapping its flat side on the glass t 22 Incubate the inocu- 21 Flame the loop and lated culture at the wire from base to tip + Actions involving the broth pure culture are in BLUE assigned temperature + Actions involving the sterile broth are in RED for the assigned time 1.15 Procedural Diagram for an Aseptic Transfer from a Nutrient Broth Pure Culture to a Sterile Nutrient Broth Tube + This is a summary of the procedure. Make every effort to keep your loop hand as still as possible throughout the transfer. Details can be found in the text. Make appropriate adjustments if transferring a BSL-2 organism.  1.17 Vortex Mixer + Bacteria may be suspended in a broth using a vortex mixer. The switch on the bottom Ill has three positions: "auto" (left), "off" (center), and "on" (right). The rubber boot is activated when touched only if the "auto" position is used; "on" means the boot is constantly vibrating. Above the on/off/auto switch is a vari- able speed knob. The slowest speed that allows the vortex to reach the bot- tom of the tube is used. Caution must be used to prevent broth from getting into the cap or losing control of the tube and causing a spill (note the hand position around the tube, ready to grab 1.16 Flaming Loop + Incineration of an inoculating loop 's it) Short bursts of vortexing can be w ire is done by passing it through the tip of the flame 's inner cone. used if the glassware is too full to allow Begin at the w ire's base and continue to the end, making sure that vortexing to the bottom. all parts are heated to a uniform orange color. Allow the w ire to cool before touching it or placing it on/in a culture. The former w ill burn you; the latter w ill cause aerosols of microorganisms. 1.19 Removing the Tube Cap + The loop is held in the domi- nant hand and the tube in the other hand. Remove the tube's cap w ith the little finger of your loop hand by pulling the tube away w ith the other hand; keep your loop hand still. Hold the cap in your little 1.18 Mixing Broth by Hand + A broth culture always should finger during the transfer. When replacing the cap, move the tube be mixed prior to transfer. Tapping the tube w ith your fingers gets back to the cap to keep your loop hand still. The replaced cap doesn't the job done safely and w ithout special equipment have to be on firmly at this time-just enough to cover the tube. 1.20 Holding the Tube at an Angle + The tube is held at an 1.21 Flaming the Tube + The tube's mouth is passed quickly angle to minimize the chance that airborne microbes w ill drop into through the flame a couple of times to sterilize the tube's lip and the it Notice that the tube's cap is held in the loop hand. surrounding air. Notice that the tube's cap is held in the loop hand. II] 7 This step is important to minimize the production of aerosols. Hold the loop hand still and move the tube up the wire until the loop's tip is in the broth. Continue holding the loop hand still while you remove the tube from over the loop (Fig. 1.22). Be careful not to catch the loop on the lip of the tube or you will produce contaminated droplets and aerosols. At this point, there should be a visible film of broth in your loop (Fig. 1.23 ). If there isn't a visible film of broth, replace the cap, flame the loop, let it cool, and then pinch the loop so it is a closed circle. Then, start over. 1.22 Move the Tube, Not the Loop + The open tube is held 8 While holding your loop hand still, flame the tube at an angle to minimize airborne contamination of it. When placing a and replace the cap. Set the tube in the rack and loop into a broth tube or removing it, keep the loop hand still and pick up the sterile broth tube in your free hand. move the tube. Be careful not to catch the loop on the tube's lip when removing it. This produces aerosols that can be dangerous 9 Repeat the process of flaming the lip of the sterile or produce contamination. broth tube while holding the loop hand still. Don't forget you have living microbes exposed to the environment on the loop at this point and careless movements can spread them. 10 Insert the loop into the sterile broth and mix by gently swirling the loop. 11 Before you remove the loop from the tube, tap the loop's face on the inside of the tube to remove the film within the loop (Fig. 1.24 ). Be persistent in this-don't give up until you are successful at its removal. If you were to flame the loop with the film present, you would produce aerosols. 12 Keeping your loop hand still, flame the tube, replace the cap, and set it in the rack. 1.23 Do Not Catch the Loop on the Tube's Lip When 13 Flame the loop from base to tip until it is uniformly Removing It + Notice the film of broth in the loop (see inset) Be careful not to catch the loop on the lip of the tube when removing orange-hot. it. This would produce aerosols and droplets that can be dangerous or produce contamination. Transfer from Agar Slant Culture to Sterile Agar Slant As you read these instructions, also follow the procedural diagram in Figure 1.25 to get a summary view of the process. This transfer has a lot in common with the broth-to-broth transfer and you are referred back to it at relevant points. Make appropriate adjustments if handling a BSL-2 organism. 1 Label the sterile agar slant with your name, the date, the medium, and the inoculum. 2 Hold the loop like a pencil and then flame it from base to tip as in a broth transfer (Fig. 1.16). 3 Loosen and remove the cap on the culture tube as in a broth transfer (Fig. 1.19). 1.24 Removing Excess Broth from Loop after Transferring + 4 Hold the culture tube on an angle with the agar Before removing it from the new culture tube, tap the face of the loop surface facing upward. on the glass to remove the broth film (Fig. 1.23). Failing to do so w ill 5 Flame the tube's lip as in a broth transfer (Fig. 1.21). result in splattering and aerosols when sterilizing the loop in a flame.  1 Label the sterile agar slant with your name, 2 Hold the loop like a pencil in your dominant 3 Flame the loop and wire from base to tip, 4 Pick up the culture slant in your free hand Ill the date, the medium, hand making sure it gets and the organism's uniformly orange-hot name t 8 Move the tube up the 7 Flame the tube's lip 6 Hold the tube on an 5 Remove the culture loop's wire until the by passing it quickly angle with the agar tube's cap with the loop is over some through the flame two surface facing upward little finger of your growth or three times loop hand + 9 Touch the sterile loop 1O Remove the tube 11 Keeping the loop 12 Return the culture to the growth on the from over the loop, hand still, flame the slant to the test tube agar surface and pick being careful not to tube's lip as before, rack up a small amount of catch the loop on and replace the cap growth the tube's lip 16 Flame the tube's lip 15 Hold the tube on 14 Remove the tube's + 13 Pick up the sterile by passing it quickly an angle with the cap with the little agar slant in your through the flame agar surface facing finger of your loop free hand two or three times upward hand t 17 Move the tube 18 Touch the agar with 19 Slowly remove the 20 Remove the tube upward over the wire the tip of the loop tube from over the completely from over so the loop is at the loop as you move the loop without base of the agar the loop in a zigzag catching the loop on pattern on the agar the tube's lip surface (do not cut the agar) t 24 Incubate the inocu- 23 Flame the loop and 22 Return the inocu- 21 Keeping the loop lated culture at the wire from base to tip, lated tube to the test hand still, flame the assigned tempera- making sure it gets tube rack tube's lip as before ture for the assigned uniformly orange-hot and replace the cap time + Actions involving the agar slant pure culture are in BLUE + Actions involving the sterile agar slant are in RED 1.25 Procedural Diagram for an Aseptic Transfer from a Nutrient Agar Slant Pure Culture to a Sterile Nutrient Agar Slant + This is a summary of the procedure. Make every effort to keep your loop hand as still as possible throughout the transfer. Details can be found in the text. Make appropriate adjustments if transferring a BSL-2 organism. II] 6 Move the culture tube up the wire of the loop, and then gently touch the loop's tip to the growth on the 10 Touch the tip of the loop to the agar where there is organism. Then, as you withdraw the tube, move agar's surface (Fig. 1.26). You don't need to dig into the loop back and forth (Fig. 1.27). Be careful not the agar, nor do you need to scoop up a glob of to cut the agar with the loop. This is called a fishtail growth. Just touch the loop to the growth and pick up inoculation or fishtail streak because you are seeding the smallest amount you can see with your naked eye. the agar surface in a wavy pattern resembling the 7 Holding your loop hand still, carefully remove the movement of a fish tail. tube from over the wire, flame the culture tube's lip, 11 Be careful not to catch the loop on the tube's lip as and replace its cap. Place the culture in the test tube you remove it. Then, keeping the loop hand still, rack. flame the tube's lip, replace its cap, and put it in the 8 Pick up the sterile agar slant, remove the cap, and test tube rack. flame the tube as before. 12 Flame the loop from base to tip as before. 9 Move the tube over the wire so the loop is near the bottom of the slant. Transfers from Plate Culture to Sterile Broth As you read these instructions, also follow the procedural diagram in Figure 1-28 to get a summary view of the process. Make appropriate adjustments if transferring a BSL-2 organism. 1 Label the sterile tube with 2 Hold the loop like a your name, the date, the pencil in your domi- medium, and the organism's nant hand name t 4A Leaving the plate on the 3 Flame the loop and table, lift the lid and use it wire from base to tip, as a shield, OR making sure it gets 1.26 A Loop and an Agar Slant + When placing a loop into a uniformly orange-hot slant tube or removing it, the loop hand is kept still while the tube is moved. Hold the tube so the agar is facing upward. To pick up the inoculum, you only need to gently touch the growth on the agar surface. t 4B With the plate inverted on 5 Touchtheloopto the table, remove the base the growth to be and hold it on an angle with transferred and obtain the agar up a small amount t 7 Continue with step 12 of 6 Keeping the loop Figure 1-15 to inoculate a hand still, replace the sterile broth or with step 13 +- plate's lid of Figure 1-25 to inoculate a sterile agar slant + Actions involving the plated pure culture are in BLUE + Actions involving the sterile tubed medium are in RED 1.28 Procedural Diagram for an Aseptic Transfer from a Nutrient Agar Plate Pure Culture to a Sterile Tubed Medium + This is a summary of the procedure. Make every effort to keep your 1.27 Fishtail Inoculation of a Slant + Begin at the base of loop hand as still as possible throughout the transfer. Details can be the slant and gently move the loop back and forth as you withdraw found in the text. Inoculation of a sterile broth is the same as in Figure the tube. Be careful not to cut the agar. After completing the transfer, 1.15, whereas inoculation of a sterile slant is the same as in Figure sterilize the loop or dispose of it properly. 1.25. Make appropriate adjustments if transferring a BSL-2 organism..I Materials 1 Flame the loop from base to tip (Fig. 1.16). 2 Lift the lid of the Petri dish and use it as a shield Per Student Ill from airborne contamination (Fig. 1.29). D Inoculating loop 3 Touch the loop to an uninoculated portion of the o Bunsen burner plate to cool it. (Loop wires can get very hot if a se- D Four sterile nutrient broth tubes ries of transfers are made in a short period of time. D Three sterile nutrient agar slants Placing a hot wire on growth may cause the growth D Marking pens and labeling tape (or materials for the to spatter and create aerosols. If you aren't doing a preferred labeling method in your laboratory) lot of successive transfers, this is probably unneces- D ( Optional) vortex mixer sary.) Gently touch the loop to some growth on the agar surface and collect the smallest amount you Per Student Group can see. As with the slant, you don't need to dig into D Nutrient agar (NA) slant culture of Staphylococcus the agar or scoop up a glob of growth. epidermidis 4 Remove the loop and replace the lid. D Nutrient broth (NB) culture of Staphylococcus epidermidis 5 Pick up the sterile broth and continue with step 9 in D Nutrient agar plate of Staphylococcus epidermidis the broth-to-broth transfer (page 34 and Fig. 1.15). In This Exercise You now have the procedures for obtaining growth from Procedure a broth culture, an agar slant culture, and a plated culture. Lab One You also have the procedures for inoculating a sterile 1 Label one nutrient broth (NB) and one nutrient broth and a sterile agar slant. These can be performed agar (NA) tube with your name and the word in any combination necessary. Today, you will begin by "sterile." testing your baseline dexterity by using sterile agar and 2 Using the sterile NB tube and the sterile NA tube, sterile broth as the "culture" tubes and the tubes to be practice making transfers between them in all possi- inoculated. After a little practice with these "blanks," ble combinations. Your lab partner has the same you will do transfers with real cultures and sterile media assignment using a second set of tubes. Work with in the following combinations: agar slant culture to ster- her/him and alternate transferring and evaluating ile agar slant and sterile broth, broth culture to sterile each other's technique. (Not only will you learn by agar slant and sterile broth, and plate culture to sterile doing and evaluating another person, alternating broth (Fig. 1.30). will allow the tubes to cool.) 3 Incubate the sterile tubes at 35 ± 2 °C until the next lab period. Source Destination Bmth Nill,iM!Bmth Slant ~ NWiMtAgfilSlant Plate 1.30 Procedural Diagram for Making Transfers of S. epidermidis + In today's lab, you will make transfers from broth and slant cultures of S. epidermidis to sterile nutrient broth and nutrient agar slant media. You will also transfer from a plate culture of S. epidermidis to a nutrient broth. 1.29 "Picking" a Colony for Transfer + Touch the tip of the loop to an isolated colony and get a small amount of growth. Use the lid as a shield from airborne contamination. II] 4 Once you have been "cleared" to make transfers us- ing real cultures, perform the following: References Barkley, W. Emmett and John H. Richardson. Chap. 29 in Methods for General and Molecular Bacteriology. Washington, DC: American a Label a sterile nutrient agar slant and a sterile nu- Society for Microbiology, 1994. trient broth with your name, the medium in the Chosewood, L. Casey and Deborah E. Wilson, eds. Page 4 in Biosafety in tube (either NA or NB), the source of inoculum Microbiological and Biomedical Laboratories, 5th ed. U.S. Department (NA slant), and the organism. Then, aseptically of Health and Human Services Publication No. (CDC) 21-1112, December 2009. transfer from the S. epidermidis NA slant culture Claus, G. William. Chap. 2 in Understanding Microbes-A Laboratory to the sterile NA slant and the sterile NB. Textbook for Microbiology. New York: W. H. Freeman and b Label a sterile NA slant and a sterile NB with Company,1989. your name, the medium in the tube, the source of Darlow, H. M. Chap. VI in Methods in Microbiology, Vol. 1. J. R. Norris inoculum (NB), and the organism. Then, transfer and D. W. Ribbins, eds. London, UK: Academic Press, Ltd., 1969. from the S. epidermidis NB to the sterile NA Emmert, Elizabeth A. B., Jeffrey Byrd, Ruth A. Gyure, Diane Hartman, and Amy White et al. " Biosafety Guidelines for handling Microorganisms slant and the sterile NB. in the Teaching Laboratory: Development and Rationale." Journal of c Label a sterile NB with your name, the medium Microbiology & Biology Education (May 2013): 78-83. DOI: http:// in the tube, the source of inoculum (NA plate), dx.doi.org/10.1128/jmbe. vl 4il.531. and the organism. Then, transfer from the S. Estridge, Barbara and Anna Reynolds. Page 712 in Basic Clinical Laboratory Techniques, 6th ed. Independence, KY: Cengage, 2012. epidermidis NA plate culture to the sterile NB. Fleming, Diane 0. Chap. 13 in Laboratory Safety-Principles and Practices, Choose a well-isolated colony and touch the 2nd ed. Diane 0. Fleming,John H. Richardson,Jerry J. Tulis, and Donald center with the loop as in Figure 1.29. Vesley, eds. Washington, DC: American Society for Microbiology, 1995. d Incubate these five tubes at 35 ± 2 °C until the Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C. next lab period. Schreckenberger, and Washington C. Winn, Jr. Chap. 2 in Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. Philadelphia: Lab Two Lippincott-Raven Publishers, 1997. 1 Remove your cultures and "sterile" practice tubes Murray, Patrick R., Ellen Jo Baron, Michael A. Pfaller, Fred C. Tenover, from the incubators and examine them for growth. and Robert H. Yolken. Manual of Clinical Microbiology, 6th ed. Washington, DC: American Society for Microbiology, 1995. Record your observations and answer the questions Power, David A. and Peggy J. McCuen. Manual of BBLTM Products and on the data sheet, page 39. Laboratory Procedures, 6th ed. Cockeysville, MD: Becton Dickinson 2 Your instructor may ask you to save your cultures Microbiology Systems, 1988. for later use. If so, put them in the refrigerator. Otherwise, remove the labels and dispose of them in the appropriate autoclave container.  Date - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Common Aseptic Transfers and Inoculation Methods OBSERVATIONS AND INTERPRETATIONS 1 Describe the appearance of growth on/in each medium. Growth on a solid medium could be described by color and amount (abundant, sparse, absent). Draw representative samples of each growth type. Growth in broth can be described by its degree of cloudiness (turbidity), using a qualitative scoring system: + + + means "very turbid" + + means "somewhat turbid" + means "barely turbid" 0 means "not turbid" Source Nutrient Broth "Sterile" practice media Staphylococcus epidermidis on NA slant Staphylococcus epidermidis in NB Staphylococcus epidermidis NA on sterile NA Plate SECTION 1 Fundamental Skills for the Microbiology Laboratory 39 -.. _r --1..r ' ,J. -·./ ~. ~·.. ) ,Jr II] QUESTIONS 1 Which medium was most difficult for you to transfer from? Which medium was most difficult for you to inoculate? Explain your difficulties. (Note: There are not correct answers to these questions. They are based on evaluation of your personal experience.) 2 Did you get growth on/in the sterile NB and NA slant tubes you practiced with? If not, congratulations. If so, where did you see it and what might have been its source(s)? 3 Did you notice a difference in density (turbidity) of growth in NB tubes inoculated from NB and NA slants? Suggest possible reasons why a difference might occur. 4 Did you notice a difference in density of growth on NA slants inoculated from NA slants and NB? Suggest possible reasons why a difference might occur.... _~ 40 MICROBIOLOGY: Laboratory Theory & Application.._ _,,,.. ' ·' '-. If#.,,  Ill Streak Plate Methods of Isolation Theory A microbial culture consisting of two or more species is separate deposited cells (CFUs) on the agar surface said to be a mixed culture, whereas a pure culture contains so individual cells (CFUs) grow into isolated colonies. only a single species. Obtaining isolation of individual A quadrant streak or a T-streak is generally used with species from a mixed sample is generally the first step in samples suspected of high cell density, whereas a identifying an organism. A commonly used isolation simple zigzag (continuous streak) pattern may be used technique is the streak plate (Fig. 1.31 ). for samples containing lower cell densities. In the streak plate method of isolation, a bacterial sample (always assumed to be a mixed culture) is streaked over the surface of a plated agar medium. During streaking, Application the cell density decreases, eventually leading to individual The identification process of an unknown microbe relies cells being deposited separately on the agar surface. Cells on obtaining a pure culture of that organism. The streak that have been sufficiently isolated will grow into colonies plate method produces individual colonies on an agar consisting only of the original cell type. Because some plate. A portion of an isolated colony then may be trans- colonies form from individual cells and others from pairs, ferred to a sterile medium to start a pure culture. chains, or clusters of cells, the term colony-forming unit Following are descriptions of streak techniques. (CFU) is a more correct description of the colony origin. Inoculation of Agar Plates Using Several patterns are used in streaking an agar plate, the Quadrant Streak Method the choice of which depends on the source of inoculum This inoculation pattern is usually performed as the initial and microbiologist's preference. Although streak patterns streak for isolation of two or more bacterial species in a range from simple to more complex, all are designed to mixed culture with suspected high cell density. 1 Obtain the sample of mixed culture with a sterile loop. 2 You have two options at this point. Use whichever is more comfortable for you or is required by your instructor. a Leave the sterile agar plate on the table and lift the lid slightly, using it as a shield from airborne contamination (Fig. 1.32). or b Place the plate lid down on the table (Fig. 1.33A). Then remove the base and hold it in the air on an angle (Fig. 1.33B). 3 Starting at the edge of the plate lightly drag the loop back and forth across the agar surface as shown in Figure 1.34A. Be careful not to cut the agar surface. The loop should contact the agar as shown in Figure 1.35. 1.31 Quadrant Streak Plate of Serratia marcescens + 4 Remove the loop and replace the lid. Note the decreasing density of growth in the four streak patterns. On this plate, isolation is first achieved in the second streak, but 5 Sterilize your loop as before. It is especially important the microbiologist wouldn't know that at the time of streaking, so to flame it from base to tip now because the loop has all four streaks are performed in the hope that isolation will occur bacteria on it. in at least one of them. Cells from an isolated colony (one that's not 6 Rotate the plate a little less than 90°. touching another colony) can be transferred to a sterile medium to start a pure culture. II] 7 Let the loop cool for a few moments (or you can touch an open part of the agar), then perform another streak with the sterile loop beginning at one end of the first streak pattern (Fig. 1.34B). Intersect the first streak only two or three times. 8 Sterilize the loop, and then repeat with a third streak beginning in the second streak (Fig. 1.34C). 9 Sterilize the loop, and then perform a fourth streak beginning in the third streak and extending into the middle of the plate. Be careful not to enter any streaks but the third (Fig. 1.34D). 10 Sterilize the loop. 11 Label the plate's base with your name, date, and 1.32 Streak Plate Inoculation: Use the Lid as a Shield + sample inoculated. The streak plate may be performed with the plate's base resting on the table while holding the lid over it to prevent airborne contamina- 12 Incubate the plate in an inverted position for the tion. Perform the streak plate as described in the text and as shown assigned time at the appropriate temperature. in Figure 1.34 or Figure 1.36. Inoculation of Agar Plates Using the T-Streak Method The T-streak method is a variation on the quadrant streak, but only three streakings are done (Fig. 1.36). There is no particular advantage of one method over the other. It basically comes down to personal preference. 1 With a marking pen, draw one line across the plate's base about one-third of the way down the plate. Then, draw a vertical line in the larger of the two regions roughly dividing it in half. The two lines make a "T." 2 Obtain the sample of mixed culture with a sterile loop. 3 You have two options at this point. Use whichever is more comfortable for you or is required by your instructor. a Leave the sterile agar plate on the table and lift the lid slightly, using it as a shield from airborne contamination (Fig. 1.32). or b Place the plate lid down on the table (Fig. 1.33A). Then remove the base and hold it in the air on an angle (Fig. 1.33B). 4 Streak the sample across the large region several times. Be careful not to cut the agar. 5 Flame the loop from base to tip and let it cool in the air or touch it to an uninoculated region of the agar. 6 Make two or three streaks out of the first region into the second region, and then continue with an addi- 1.33 Streak Plate Inoculation: Plate Upside Down + tional four or five streaks exclusively in the second (A) Some microbiologists prefer to hold the Petri dish in the air when performing a streak plate. To do this, place the plate lid reg10n. down on the table and lift the base from it, holding it on an angle. 7 Flame the loop from base to tip and let it cool in the (B) Perform the streak as described in the text and as shown in air or touch it to an uninoculated region of the agar. Figures 1.34 and 1.36.  Ill 1.34A Beginning the 1.34B Second Streak +