Microbiology Labortary Excercises, Exercises of Biochemistry

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MICROBIOLOGY

Laboratory Exercises

Microbiology

Laboratory Exercises

Third Edition

Ramaydalis Keddis, Ph.D. Ines Rauschenbach, Ph.D.

Department of Biochemistry and Microbiology Rutgers, The State University of New Jersey

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LABORATORY SCHEDULE

WEEK TOPIC

1 Biosafety; Best Lab Practices; LabArchives Notebook Preparation of a culture medium Culturing and handling microorganisms

2 Follow Up: Culturing and handling microorganisms Isolation of a Pure Culture Counting bacterial populations: Plate counts

3 Follow Up: Isolation of a Pure Culture Follow Up: Counting Bacterial Populations Control Of Microorganisms – Experimental Planning

4 Microscopy – Introduction Control Of Microorganisms – Lab Work

5 Microscopy – Phase Contrast (Yeast and Bacteria) Control of Microorganisms (Poster Work)

6 Microscopy – Phase Contrast (Unknowns) Control of Microorganisms (Poster Work) Selection of Fungi or Bacteria

7 Follow Up: Selection of Fungi or Bacteria Microbial Staining Control of Microorganisms (Poster Work)

8 Microbial Metabolism: Enzymes, Metabolic pathways Control of Microorganisms (Finish Poster)

9 Follow Up: Microbial Metabolism Poster Presentations

10 Transformation Plasmid Isolation Poster Presentations

11 Follow Up: Transformation Microbial Genetics: Electrophoretic analysis of restriction digest products (Plasmid Isolation)

12 Lab Practical

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Best Laboratory Practices The organisms used in General Microbiology are classified as Risk Level 1 organisms by the ABSA ( American Biological Safety Association ) as they are not known to cause disease in healthy humans. You will learn the techniques of safe microbiology using risk level 1 organisms; however, we will maintain bio safety 2 standards when working with all organisms. The following list of best practices is printed here to remind you of the safety discussion presented during the first laboratory meeting.

x Do not bring food or drinks into the laboratory. x No sandals – toes must be covered. x No shorts or short skirts. Long pants or skirts must be worn at all times. x Cell phones and other non-lab electronic devices are not permitted. x Lab coats and safety glasses are required. x Long hair must be tied back. x Always use aseptic technique when working with organisms in culture. x Wash your hands before leaving the laboratory. x Place coats, books, and other personal possessions in assigned space. x Cleanliness: Before beginning your laboratory work, wipe down the benchtop with a microbiocide (e.g. Konflict) x Instructions for disposal will be provided during your lab period. Never throw anything down the drain. The following will help when you are unsure.

General Instructions for disposal: x Microscope slides and cover slips go in the red “sharps” containers. x Pour excess stains into labeled hazardous waste containers. x Broken glass should be disposed off in the broken glass container (Figure I-1).

Figure I-1. Receptacle for broken glass.

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Figure I- 3 Common Equipment Used in the Microbiology Laboratory.

A Biosafety Cabinet with Incinerator E Shaker incubator B Water Bath F Incubator C Electrophoresis gel box and power supply G Light microscope D Centrifuge

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(1) PREPARATION OF A CULTURE MEDIUM

The study of microorganisms in the laboratory generally requires growing organisms on a nutrient medium. Culture media must meet the nutritional requirements of the organism you wish to culture. Minimal nutritional considerations include the macronutrients; carbon, nitrogen, and phosphorus, and various other minerals, such as iron and magnesium, in lesser amounts. In addition, most microbiological media incorporate buffers, weak acids or bases that resist changes in pH. Of course, all living organisms require water.

Many different types and formulations of media are used for growing bacteria and fungi in pure culture. There are also media specific for the selection and differentiation of a vast variety organisms. For our routine laboratory work, a complex medium (exact composition is not known) prepared from plant, fungal and/or animal extracts is appropriate to meet the nutritional needs of most of the organisms we study. Many different types of media are available commercially as pre-formulated dry powders to be reconstituted in the laboratory. Appendix C at the end of this manual describes all media used in General Microbiology.

A broth medium is one in which the ingredients are simply dissolved in water; a solid medium is usually prepared with agar, a seaweed polysaccharide that acts as a gelling agent. Agar is a superior gelling agent in microbiological media because of its unique physical properties. Once gelled, agar remains a solid up to 90°C (gelatin melts at 35°C). Solid agar does not melt until it is heated to 100°C; liquid agar does not solidify until it is cooled to about 42°C allowing liquid agar to be poured into Petri plates at a temperature relatively easy to handle (Figure 2.1). For most microorganisms, agar is an ingredient which they cannot use as a nutrient.

The media prepared in this exercise will be sterilized to kill microorganisms introduced during preparation and used in subsequent experiments during the course. Growth media are routinely sterilized in the autoclave by heating at 121°C under pressure (15 psi) for 15 minutes (Fig.1.1). The autoclave is also necessary, as discussed in the safety training, to kill cultures after use.

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(2) CULTURING AND HANDLING MICROORGANISMS

Basic laboratory techniques in microbiology must be mastered not only by the microbiologists, but also by molecular biologists, geneticists, biochemists and many other scientists, as these techniques are used as tools in many disciplines. These techniques include the transfer of microbial cells from one medium to another without introducing extraneous species from the environment or contaminating the in a variety being studied (aseptic technique); of formats. The preparation of culture media that will support the growth of the microorganisms being studied; and the isolation of pure cultures from mixed populations and their maintenance in the laboratory (Figure 2.1 and Table 2.1).

Table 2.1 Overview of media formats.

Media type Description

Liquid Medium

Broth Any liquid medium dispensed in a tube or flask, used for growth of a variety of organisms

Solid Media

Deep Provides little surface for growth, must be stabbed for inoculation; used for testing oxygen limitation on bacterial growth

Slant Maintenance and storage of pure cultures

Plate Provides a large surface area for growing bacteria but dries out fairly rapidly; commonly used for the isolation of individual colonies

In this exercise you will accomplish aseptic culture transfer. Aseptic technique requires working in a different way; for most students, General Microbiology Lab is the first time they have worked aseptically in the lab. The cardinal rule for working aseptically is – Don’t open anything until the instant you need it & and don’t leave it open longer than absolutely necessary. You will use a sterile inoculating needle to transfer cells from a pure bacterial culture to the surface of an agar slant, establishing a fresh culture of cells on a new culture medium.

Figure 2.1 Assortment of Media

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You should be able to accomplish this simple task without introducing, into either the old or the new culture, contaminating species of bacteria or fungi. Aseptic technique also protects the work space and environment around the workspace from contamination by the organism being transferred.

MATERIALS Various slant cultures

1 TSA slant per student, Bunsen burner/Striker, Test tube rack, Inoculating needle

PROCEDURE

There are numerous methods to accomplish the transfer of microorganisms. The following method is generally easiest for beginning students. Before you get started, be sure to watch the microlab clip on Canvas about culture transfer. Whenever you make an aseptic transfer in lab you will use a Bunsen burner. The flame of the Bunsen burner is used to sterilize equipment and also provides convection currents that keep the air near your work free of spores and bacteria. You should always work near your own burner flame for this reason as well as the safety concerns of reaching from a distance to flame your needle.

1. Sterilize the inoculating needle in the flame of a Bunsen burner. Hold the needle nearly vertically and heat the entire length of the wire portion of the needle until it glows red. Allow 10 seconds (count to 10 slowly or quickly to 20) for the wire to cool before proceeding. Do not wave the needle around in the air to cool.
2. Hold the culture to be transferred in the left hand (for a right-handed person) and remove the cap using the last two fingers of the right hand, the same hand in which you are holding the sterile needle. Never put the cap down on the bench. Note: We do not flame the open tube either before or after the culture sample is removed. This flaming is historic, a reflection of times when cotton plugs were used (Figure 2.2). There is still debate on this topic (1)
3. Insert the needle and touch the medium to ensure that it is cool, then with the needle, remove a small amount of the surface growth from the slant. Do not gouge the agar. Replace the cap on the culture tube and put it back into the test tube rack. Pick up the fresh agar slant and follow the same procedure to aseptically remove the cap from the tube.
4. Evenly streak the surface of the new slant from the bottom upward, with a back and forth motion. Replace the cap on the tube and flame the inoculating needle to kill any residual inoculum before putting it down.
5. Label this tube appropriately and make sure to record the name of the culture transferred in your notebook. Incubate the culture @ 35°C. The culture will be removed from the incubator after ~24 hrs. and refrigerated until the next laboratory period.

WEEK TWO

1. Examine the growth on your slant macroscopically. Describe any growth in your notebook. Do you assess your culture transfer to be successful?

Figure 2.2 Cotton-Plugged Tubes

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To obtain a pure culture, cells are picked from an isolated colony with an inoculating needle and transferred to a fresh culture medium. Cells that grow on the new medium are a subculture of the cells from the original isolated colony. Subcultures are used for subsequent study of the isolated organism. Contaminating organisms from the original culture remain on the streak plate.

In this exercise you will use the quadrant streak plate technique to isolate a pure culture from a mixed culture containing four different bacterial species. The culture that you isolate and establish as a pure culture will be used in later exercises to demonstrate methods for identifying unknown microorganisms.

MATERIALS

Week 1 A mixed broth culture containing three to four unknown organisms One plate of TSA, Bunsen burner/Striker, Test tube rack, Inoculating loop

Week 2 1 TSA slant per student, Bunsen burner/Striker, Test tube rack, Inoculating needle

PROCEDURE – WEEK ONE

1. Use your sharpie and divide your Petri dish into four quadrants. Number each quadrant 1 – 4. (Figure 3.3) Label your Petri dish. Please be sure to also review the micro lab clip on Canvas about the proper QSP procedure.
2. Sterilize the inoculating loop in the flame of the Bunsen burner and cool. Either quickly count to 20 or slowly to 10. Remove a loopful of the mixed culture to the agar surface and inoculate this area with a continuous back and forth motion as if you were trying to rub off all of the cells clinging to the loop.
3. Flame the loop to sterilize and cool. Touch Figure 3.3 The quadrant streak plate the loop to a clean spot on the agar plate method. to assure that it is cool. Streak through the area in the inoculated sector and into the next sector four times. Use straight lines making sure to lift the needle before each subsequent streak line.
4. Repeat as in step 3 until the four sectors on the plate have been inoculated. The idea is to streak so that each succeeding sector contains fewer cells than the preceding one, resulting in at least one sector with cells spaced far enough apart to allow isolated colonies to develop.
5. Label the agar side of the plate; incubate the cultures, agar side up, @ 35°C. The plates will be removed from the incubator after 24 hrs and refrigerated until the next laboratory period.

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4 3

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WEEK TWO

1. Examine the pattern of growth to confirm that the procedure was followed correctly, and that isolated colonies were obtained in one of the sectors. If you failed to get isolated colonies, streak another plate.
2. Transfer cells from an isolated colony to the agar slant provided. Incubate @ 35 °C for 24 hrs and refrigerate until the next laboratory period. Save your slant in the refrigerator since it will be used as your unknown organism for identification in later exercises. Save the original plate (in the refrigerator) until the next class period.

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3. Label the bottom of the Petri dishes with your name and the dilution to be counted. Dispense the appropriate volume (1.0 ml) of the dilutions you are counting into the bottom of sterile Petri dishes. Aseptically, dispense agar (melted and held at 50°C in the incubator) into the Petri dish as follows. Anytime you are working aseptically, you need to work near a Bunsen burner. Never share a Bunsen burner with someone else, as this will not allow you to work close enough to the burner to take advantage of the convection currents it generates.
4. Before you start, make sure to watch the micro lab clip on Canvas to see the demonstration of the technique. The procedure is as follows: lift the cover of a Petri dish and aseptically pour the liquefied agar into the bottom section of the dish. Replace the lid as soon as the agar has been poured. Repeat the procedure with the second medium. Allow about 5 to 8 minutes for the agar to solidify.
5. Gently mix the agar with the dilution sample by swirling gently in a “Figure 8” pattern as demonstrated by your instructor, and let the agar harden. Incubate the cultures by placing the plates, agar side up, in the bins provided. The cultures will be incubated at room temperature for three days and refrigerated until the next lab period.

WEEK TWO

1. To determine the number of cells in the original culture, select the plate that has between 30 and 300 colonies; multiply the number of colonies by the dilution factor for that plate. Plates with more than 300 colonies are recorded as Too Numerous To Count (TNTC).

Figure 4.1 Dilution Plates. A: Successful dilution series; B: Unsuccessful, the culture was not mixed properly before the agar solidified; C: Agar was not distributed properly in the Petri dish before it solidified.

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(5) CONTROLLING MICROBIAL GROWTH

We will be working in the groups for this exercise. Each group will select one of the following microbial control agents: antiseptics, disinfectants, antibiotics, hand washing or UV light. As a group, formulate an original testable hypothesis, using the methods in the manual as a guide to design your experiment. Groups will perform the experiment (make sure you include controls) deciding on incubation temperature and time, examine results and draw conclusions as a group. The collected information will then be compiled in a poster and presented at the end of the semester.

CHEMICAL METHODS TO CONTROL MICROBIAL GROWTH

5 - 1 DISINFECTANTS AND ANTISEPTICS

Chemicals that kill microorganisms or prevent the growth of microorganisms are called antimicrobial agents. They are widely used in various industries including, for example, health care settings and food service. Antimicrobial agents are found in a broad range of consumer products from mouthwash to cereal boxes and beyond. Concentration and contact time are the critical factors determining the effectiveness of an antimicrobial agent against a given microorganism. Microorganisms vary in their sensitivity to antimicrobial agents. The activity of many antimicrobial agents is aimed at blocking active metabolism and preventing the organism from generating the macromolecular constituents needed for reproduction. Because resting stages such as spores are metabolically dormant, they are not affected by such antimicrobial agents. Similarly, viruses are more resistant than other microorganisms to antimicrobial agents because they are also metabolically dormant outside host cells.

Antimicrobial agents are used in a wide variety of applications and are classified according to their application and spectrum of action. Germicides are chemical agents that kill microorganisms. Such chemicals may exhibit selective toxicity. Depending on the target organisms they are classified as viricides (viruses), bactericides (bacteria), algicides (algae), or fungicides (fungi). Whereas germicides kill growing microorganisms, “static” agents inhibit the growth of microorganisms but do not kill them i.e. when a bacteriostatic agent is removed, bacterial growth resumes.

Disinfectants are used on surfaces and equipment but not biological tissue. They can be either the germicidal or germistatic agents. Household cleaning agents often contain disinfectants to control the growth of microorganisms. Ammonia and bleach (hypochlorite) are widely used disinfectants. In general, the agents that oxidize biological macromolecules such as hypochlorite are effective disinfectants. Antiseptics are similar to disinfectants but may be applied safely to biological tissues. These substances are used for topical (surface) applications (e.g. on the skin) but are not necessarily safe for consumption. Alcohol (70%) is effective in reducing the numbers of microorganisms on the skin surface and is probably the most widely used antiseptic. It may be used on the skin in the area of a wound as well as for the disinfection of various contaminated objects. Alcohol may denature proteins, extract membrane lipids, and/or act as a dehydrating agent, contributing to its effectiveness as an antiseptic. Even viruses are inactivated by alcohol. Soap

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2. Label the agar side of the Petri plates with the name of the microorganism being tested. Indicate which substance is being evaluated for each disk placed on the agar surface.
3. Using sterile forceps, dip a sterile filter disk into one of the solutions provided and place the saturated disk onto one of the inoculated agar plates. (If there is an excess of solution on the filter paper disk, drain it on a paper towel prior to placing on the agar.)
4. Repeat this procedure for all solutions being tested.
5. Incubate the cultures for 24-48 hr.

WEEK TWO

1. Examine the plates for clear areas (zones of inhibition) in the lawn of confluent bacterial growth surrounding the disks. Measure and record the sizes of the zones of inhibition.

Figure 5. 2 Example plate showing zones of inhibition.

5 - 2 ANTIMICROBIAL AGENTS USED IN VIVO : SUSCEPTIBILITY TESTING

Resistance of microbes to the effects of antimicrobial agents is a serious problem in the world today. In 2014, the World Health Organization Global Surveillance Report (2) stated the problem is “so serious that it threatens the achievements of modern medicine. A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.” Monitoring the susceptibility/resistance of microbes has become critically important.

The Kirby-Bauer method (3) is the classical method for determining the susceptibility of an organism to an antimicrobial agent. An agar plate is inoculated with the culture to be evaluated. The antimicrobial agent is allowed to diffuse into the agar medium, usually from a filter paper disc. The entire surface of a Petri dish is swab inoculated with the test organism to create a “lawn” of growth. A filter paper disk impregnated with an antimicrobial agent is applied to the surface of the inoculated agar plate. The antibiotic diffuses radially from the round filter paper into the agar: the concentration decreases as a function of the square of the distance from the disk. At some distance from each disk, the antimicrobial agent is diluted to the point that it no longer inhibits microbial growth. The effectiveness of a particular antimicrobial evidenced by the presence of growth inhibition-zones (Fig 5.3). These

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zones of inhibition appear as clear areas surrounding the disks. The diameter of the zones is measured, and the relative efficacy of the antimicrobial agent is determined by comparing measured zone sizes to standards. The relative effectiveness of different antibiotics provides the basis for the sensitivity spectrum of the organism. The disk diffusion method represents a simple procedure for screening substances to determine if they have significant antimicrobial activity.

The relative sensitivity of the organism is one factor considered in selecting antimicrobial agents for treatment. A number of experimental variables including viscosity of the culture medium, solubility of the antimicrobial agent etc. influence the size of the clearing zone. Many antimicrobial compounds produce adverse effects that must be considered when prescribing chemotherapeutic agents.

In this exercise, you will test the susceptibility of bacteria to a number of antimicrobial agents. Although we tend to think of antibacterial agents as antibiotics, this is not the proper terminology. In 1947, Selman Waksman (4) defined antibiotics as “chemical substances that are produced by microorganisms and that have the capacity, in dilute solution, to selectively inhibit the growth of and even to destroy other microorganisms.” Antibiotics are then, by definition, natural products affecting any other microbe. The term “antimicrobial agent” broadly refers to any compound, natural or synthetic, that inhibits the growth of a microbe (bacterial, fungal, viral, protozoal etc.) Please use these terms correctly.

MATERIALS Cultures of your choice Filter disks containing a number of different antimicrobial agents/various dosages Ethanol Mueller Hinton Agar Plates, 150 mm in diameter Ruler graduated in millimeters Tweezers

PROCEDURE – WEEK 1

1. Uniformly “swab inoculate” the entire surface of the Petri plates with one of the broth cultures. This may be accomplished by swabbing to cover one-half of the plate area, rotating the plate 90°, swabbing over the initial inoculum with the same swab and covering one-half of the plate area, rotate the plate 90°, following the same procedure, and rotate the plate one more time repeating procedure. You will be swabbing 4 different times.
2. Aseptically place antimicrobial disks on the culture plates. Using sterile forceps place the antimicrobial disks on the surface of the agar plate inoculated with the test organism; space them in equal sectors such that no more than 8 disks are placed on a plate.
3. Incubate at 30°C for 24h.

WEEK TWO

1. Measure the zones of inhibition for each antimicrobial agent for each of the cultures. Record your results.
2. Compare your results with standards (Thermo Fisher for product insert from the Clinical Laboratory Standards Institute document M100-S23) provided for each antimicrobial agent and determine the sensitivity, resistance, or intermediary relationship of each organism to each antimicrobial agent.