BIOL2038 MICROBIOLOGY

Practical 2 Schedule
2011/12 Semester 2

Name: Group A/B:

Lab Partner:

Demonstrator/ Assessor
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Unless certain precautions are taken laboratories can be hazardous places so make sure you have read the guidelines below. All bags and personal belongings should be placed in the lockers outside the lab before starting work. All food, drink and mobile phones should remain in your bag. The only exceptions are items required for the practical and its write-up or any medication that may be required in a hurry. Always wear a laboratory coat when performing experiments in a laboratory. This simple precaution may save you from serious burns and will certainly extend the life of your everyday clothing. Laboratory coats should be removed when leaving the laboratory and never taken to areas where food and drink are dispensed. Laboratory coats will not protect you or your clothing unless they are kept buttoned up. Wear safety equipment when instructed to do so by the demonstrators or members of staff. If safety goggles and/or gloves are provided as part of the experimental apparatus it is sensible to use them. If you are not sure, make the assumption that all chemicals you use in the laboratory are toxic, corrosive and carcinogenic. In particular, the vapours of organic solvents must be considered dangerous and should be used, where possible, in a fume cupboard or at least in a well ventilated room After completion of an experiment check with the demonstrator about the correct way to dispose of any waste materials. Some substances cannot be put down the sink and some items cannot be put into normal bins. Always wash your hands before leaving the lab. Personal hygiene will greatly assist in the prevention of poisoning and disease and will also help prevent such unpleasant conditions as dermatitis.

Above all, take care and pay attention. It is very easy to knock things over with a careless swing of the arm, or to endanger others through a lack of awareness of your surroundings. If the guidelines above are followed the practicals are safe to perform and there shouldnt be any need to follow the emergency information on the following page. ACTION IN EVENT OF ACCIDENT In the event of an accident involving personal injury, contact any staff member or a qualified First Aider (see below). If necessary, emergency services should also be contacted as soon as possible. Any accident involving personal injury must be reported to a member of the Academic Staff and an accident report made. In the event of a potentially serious incident, even if no injury occurs, a report should be made to the appropriate safety officer so that action can be taken to prevent future, possibly serious, accidents taking place.
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ACTION IN CASE OF FIRE IF YOU DISCOVER A FIRE Raise the alarm and call for assistance. Attack the fire with the nearest suitable appliances only if it is possible without endangering yourself. Dial 112 and ask for 'fire brigade'. Be prepared to detail the location of the fire. IF YOU HEAR THE ALARM Close all doors and windows, where possible. If possible without delaying departure, ensure that other persons in the vicinity are aware of the alarm. Leave the building by the nearest available exit. Get well clear of the building. (Assembly point - CAR PARK) DO NOT RUN - panic is infectious Do not return for any reason unless told that it is safe to do so. Please take the fire drills seriously. You should be familiar with the building so that you know the positioning of escape routes, fire extinguishers and the alarm bells.

SAFETY STAFF Safety Officer: First Aiders: Mr Neville Wright Room; Tel: 24306 Mr David Norris Tel: 22027 Mr Nick Orson Tel: 22027

University Medical Officer Ext 23539/23739 (daytime) Ext 557531 (out of hours after 5pm weekdays) University Maintenance Division Ext 22811

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The media on which you culture microorganisms will readily grow undesirable contaminants, for example molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures and yourselves from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact. Spray and wipe down your bench surface with 70% ethanol before starting experiments and at the end of the lab. This will help to keep your work area free of contaminating organisms. Where possible work closely to the roaring flame of a Bunsen burner the sterile air surrounding the burner will help to protect your experiment from contamination. Never leave a culture dish open, even for a short time when viewing colonies of organisms, unless you intend to discard it. When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface. Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking off the cap. Hold the cap with opening down, and the tube horizontal or nearly so. Convection from the heated neck will prevent dust from falling into the opening. Flame again before putting the cap back. Use sterile disposable pipets to remove samples from a broth culture that must be kept uncontaminated. Always be aware of where your hands are, where your face is, and whether or not your culture is in a position to be contaminated. If you have long hair, make sure it does not hang into your plate. Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms. If you have an open flame, long hair that is not tied back or loose clothing can be hazardous. Keep flammables away from the flames, including alcohol used for sterilizing instruments; do not place a heated loop or glass rod into an alcohol dish Follow these two basic principles: Do not allow micro-organisms to escape from culture vessels Do not allow contaminating organisms to enter the vessel Other general rules: Work quickly, tidily and methodically
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Set out your apparatus in a convenient arrangement, so that you need move only short distances Do not carry pipettes loaded with culture across the laboratory Place contaminated pipettes into the disinfectant provided When a pure culture is to be maintained, you must make sure our apparatus and solutions are sterile Never leave cultures open to the atmosphere for a longer time than is necessary When opening cultures, always incline the tube or flask away from the vertical so that airborne contaminants cannot fall in Always kill cultures with disinfectant before discarding them Swab any accidentally spilled culture with disinfectant and notify a demonstrator Treat all micro-organisms as actual or potential pathogens

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Microscopy Quick Reference Sheet

Notes: If you use immersion oil, ONLY use the OIL labelled lens, and wipe it clean after use with lens tissue provided. If you wear glasses, you may find it easier to push back the glare protection, as it may prevent you seeing the whole field of view. PLEASE MAKE SURE you email ALL images you acquire with the microscope to yourself and your group as they will be DELETED on log out.
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REFRESHER - Method for Setting up compound microscope for bright field.

Follow the instructions below. Remember, if you have any problems at all, ASK A DEMONSTRATOR. a. Before looking through the microscope: 1. Refer to the labelled diagrams of microscope 2. Familiarise yourself with the parts of the microscope (the diagrams should help). Insert the plug into an electric socket. Make sure the mains switch (on the side of the base unit) is switched off. The light intensity control (rheostat) must be turned down to minimum. Turn on the power at the socket and then at the base unit and increase the light intensity slowly 3. Using the condenser focusing knob, move the condenser into roughly the correct position - just below the top surface of the stage (raise it as high as it will go then lower it by about 1 mm). 4. Move the low power objective (x10 or x4) into its correct operating position 5. Place a specimen on a slide with a drop of water, place a cover slip over the specimen, put the slide on the stage securing with the spring clip and centre it (you will be shown how to do this). 6. Turn up the stage to the top position b. Now look through the microscope 1. If you have a binocular eyepiece adjust the separation (interpupillary adjustment /oculars) so that you can comfortably see a single circular field of view while observing the image with both eyes 2. Focus on the specimen using coarse then fine focus knob 3. To compensate for unequal eye accommodation, focus the image of the specimen using the right eye alone (AND THE RIGHT (FIXED) EYEPIECE!). Next, without touching the focus controls, refocus the image with the other eye alone by rotating the dioptre adjustment (milled ring on the adjustable eyepiece tube). 4. Focus the specimen sharply, close the field diaphragm then focus the condenser (using the knob under the stage) until the edge of the field diaphragm is sharply focused. Open the diaphragm until it meets the edge of the field of view. If it is not centred adjust it by moving the field diaphragm centreing screws (attached to the light source turret at an angle to the sides). In the Swift the lens can be pushed into the right position. Open the diaphragm a little further until it is no longer visible
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5. The light intensity should not be altered by means of the condenser iris; it should be adjusted by means of the light intensity control (rheostat) 6. If you need higher magnification then turn the objectives round (the magnification is printed on individual objectives.). Be careful not to move the slide and use fine NOT coarse focus controls. 7. Practise the steps above until they become second nature. Test your partner (several times if necessary). We may test your expertise with a spot test!

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PRACTICAL 2

MICROSCOPIC OBSERVATION OF BACTERIAL CELLS Aims

1) To analyse data obtained from the previous practical, including observation of streak plates and recording serial dilution plate counts and bacterial transformation data, 2) To carry out the Gram Stain method for the microscopic observation of Gram positive and Gram negative bacteria, 3) to carry out observation of GFP fluorescent bacterial cells under epifluorescence illumination, 4) To inoculate E. coli biofilm cultures in preparation for Practical 3 Investigation of antibiotic tolerance in planktonic v biofilm culture. Skills: Gram staining technique, light field and epifluorescence microscopy, analysis and interpretation of data from experiments and controls, biofilm culture.

1. Data collection from previous practical.

Materials for Student pairs Materials from Practical 1, Refrigerated streak plates, dilution series plates, and transformation and control plates (LB/amp/ara 1, LB/amp 2, LB 1). Common equipment UV lamp/s

Streak plates. Examine your plates to see whether you have been successful in isolating single E. coli bacterial colonies. Also how good was your aseptic technique are there any contaminating colonies on the plates?! If so, these could be fungi or other bacteria from the air or surfaces that have contacted your experiment.

Dilution Series - Record and count the number of bacteria on each of the plates from your dilution series. NB Count only plates that have between 20 and 300 colonies. Hopefully, if your technique was good, you will see approximately 10-fold differences in the number of bacterial colonies between dilutions. You will need your counts to calculate the number of bacteria ml-1 in the original bacterial suspension (see Analysis section at end of report).

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pGLO GFP transformation data Observe the results of the transformation experiment under both normal and UV lighting and record the information as detailed below. You will need this data for calculations of transformation efficiency (see Analysis section at end of report).

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2. Gram Stain.
Materials for Student pairs Bunsen burner Microbiological waste disposal bin 70% ethanol to spray/sterilise bench Blue tissue roll to wipe bench 1:1 Mixture of E. coli and Listeria innocua o/n broth culture (1 ml) Microscope slides (box) Cover slips (box) Immersion oil Sterile inoculation loops Pipettes (200 ul) and tips Primary Stain: Crystal Violet Staining Reagent Mordant: Gram's Iodine Decolorizing Agent (Ethanol, 95%) Counterstain: Safranin Slide holder/Tray Background. The Gram stain was first used in 1884 by Hans Christian Gram. It is fundamental to the phenotypic characterization of bacteria. The staining procedure differentiates organisms of the domain Bacteria according to cell wall structure. Gram-positive cells have a thick peptidoglycan layer and stain blue to purple. Gram-negative cells have a thin peptidoglycan layer and stain red to pink. Theory. The Gram stain, the most widely used staining procedure in bacteriology, is a differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain purple. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content. These stain pink. This staining procedure is not used for Archeae or Eukaryotes as both lack peptidoglycan. The performance of the Gram Stain on any sample requires four basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Grams Iodine), rapid decolorization with alcohol, and lastly, counterstaining with safranin. The CV interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV to form large CVI complexes within the cytoplasm and outer layers of the cell. The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell. The highly cross-linked and multi-layered
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peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell. After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Gram positive and Gram negative cell wall composition

Protocol 1. Transfer a drop (10 20 L) of bacterial suspension and spread over the centre of a microscope slide. If staining from solid media add a drop of sterile distilled water to slide and mix in a small amount of a colony. Allow to air dry then fix cells by passing through a gentle flame (move the slide in a circular fashion to avoid overheating) When cool flood the slide with Grams crystal violet stain. Allow to stand for 60 seconds. Pour off the stain and wash gently with distilled water or in a gentle and indirect stream of tap water for 2 seconds Flood with Grams iodine solution. Allow to stand for 60 seconds. Pour off the stain and wash gently with water. Shake off excess water. Decolourise with decolourising solution until blue dye no longer flows from the smear (95% ethanol). Be careful not to over-decolourise or Gram-positive cells could appear to be Gram-negative. Wash slide with water. Counterstain with Grams safranin solution for 60 seconds.

2. 3. 4. 5. 6. 7.

8. 9.

10. Wash slide with water, shake off excess water (can blot edge of slide with absorbent paper) and allow to air dry.
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11. Place a drop of immersion oil on the dried smear and examine microscopically (oil immersion objective): Gram-positive cells appear purple ; Gram-negative cells appear pink.

3. Microscopic Observation of pGLO GFP fluorescent cells

Materials for Student pairs Eppendorf containing 1.0 ml sterile water or PBS Inoculation loops Dropper Microscope slide (box) Coverslip (box) Immersion oil Communal equipment Fluorescence microscopes Protocol Make sure that you have identified a GFP-fluorescent colony from your transformation plates (use a marker pen on the base of the Petri dish after viewing under UV light if necessary). Place a small drop (10 L) of sterile distilled water on the slide and mix in a VERY SMALL amount of a colony. Gently place a cover slip over the bacterial suspension and examine under fluorescence microscopy (see appendix 1 fluorescence microscopy) for further information and theory on epifluorescence microscopy). GFP fluoresces under blue light illumination view at x 1000 magnification if possible.

4. Investigation of antibiotic tolerance in bacterial biofilms vs. planktonic cells. Pt. 1: Biofilm Culture Inoculation
Background: One of the most important properties of biofilms is the increased tolerance to antimicrobial substances, both disinfectants and antibiotics. This tolerance has implications for both clinical and industrial microbiology. This experiment will form the majority of your write-up and will utilize methods for determining the resistance of biofilms to antibiotics and for comparing that resistance to planktonic cells of the same species. In this practical you will start a biofilm culture for investigation in Practical 3.
Materials for Student pairs E.coli o/n broth starter culture (aliquot of 10 mls) Pipette and 1ml tips Sterile 6-well plate
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Cover slips (box) Metal tweezers Absolute acetone (15 ml in 50 ml Falcon tube) Absolute Ethanol (15 ml in 50 ml Falcon tube) Sterile 1/10 strength LB broth 50ml Pipettes and tips

Protocol:
Preparation of the coupons and biofilm inoculation 1. Dip the cover slips (glass coupons) in absolute acetone and then in absolute ethanol. 2. Remove the excess of ethanol, flame and place in a 6-well plate. 3. Place 6 cover slips, one in each well, into a 6-well plate. 4. Pipette 4.5 ml of 1/10 LB to each well containing the SS coupons. 5. Add 500 l of the overnight inoculum to each well. 6. Incubate at 37C for 24 48 hours (Plates will be then be placed in the fridge for 2 weeks for the next practical.)

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ANALYSIS (Important This section to be submitted as an appendix to your final experimental write-up)
1. Serial dilution. Report the number of colonies on your serial dilution plates and calculate the concentration of bacteria in the original unknown bacterial suspension (see notes from Practical 1 schedule on how to do this calculation). Pay attention to units and correct scientific notation for powered numbers etc.

2. PGLO GFP Transformation data The goal of data analysis for this investigation is to determine the extent to which genetic transformation has occurred. 1. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the genes on the plasmid that you used in your transformation procedure?

2. From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed?

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Whats Glowing? If a fluorescent green color is observed in the E. coli colonies then a new question might well be raised, What are the two possible sources of fluorescence within the colonies when exposed to UV light? Explain: 1. Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observations.

2. Which of the two possible sources of the fluorescence can now be eliminated?

3. What does this observation indicate about the source of the fluorescence?

4. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.

The Interaction between Genes and Environment Look again at your four plates. Do you observe some E. coli growing on the LB plate that does not contain ampicillin or arabinose? 1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.

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2. How would you change the bacterias environmentthe plate they are growing onto best tell if they are ampicillin resistant?

3. Very often an organisms traits are caused by a combination of its genes and its environment. Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacterias environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria).

b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria to turn green?

c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

Transformation Efficiency Calculation Your next task is to determine the extent to which you genetically transformed E. coli cells. This quantitative measurement is referred to as the transformation efficiency and gives you an indication of how effective you were in getting DNA molecules into bacterial cells. It represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The transformation efficiency is calculated using the following formula:

Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate (in g) Therefore, before you can calculate the efficiency of your transformation, you will need two pieces of information: (1) The total number of green fluorescent colonies growing on your LB/amp/ara plate. ENTER that number here:

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(2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate as follows: In this experiment you used 10 l of pGLO at concentration of 0.08 g/l. Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread onto the LB/amp/ara plate: Since not all the DNA you added to the bacterial cells will be transferred to the agar plate, you need to find out what fraction of the DNA was actually spread onto the LB/amp/ara plate. To do this, divide the volume of DNA you spread on the LB/amp/ara plate by the total volume of liquid in the test tube containing the DNA. A formula for this statement is Fraction of DNA used = Volume spread on LB/amp plate (l) Total sample volume in test tube (l)

You spread 100 l of cells containing DNA from a test tube containing a total volume of 510 l of solution. Use the above formula to calculate the fraction of pGLO plasmid DNA you spread on the LB/amp/ara plate. RECORD THAT NUMBER HERE: Fraction of DNA =

So, how many micrograms of pGLO DNA did you spread on the LB/amp/ara plates? To answer this question, you will need to multiply the total amount of pGLO DNA used in this experiment by the fraction of pGLO DNA you spread on the LB/amp/ara plate. pGLO DNA spread in g = Total amount of DNA used in g x fraction of DNA used RECORD THAT NUMBER HERE: pGLO DNA spread (g) =

Now calculate the efficiency of the pGLO transformation :

Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate RECORD THAT NUMBER (AND CORRECT UNITS) HERE:

Transformation Efficiency =

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Analysis Ctd. ADDITIONAL QUESTIONS: 1) Calculate the transformation efficiency of this example experiment using the information and the results listed below. DNA plasmid concentration: 0.08 g/l 250 l CaCl2 transformation solution 10 l pGLO plasmid solution 250 l LB broth medium 100 l cells spread on agar 227 colonies of transformants

2) If a particular experiment were known to have a transformation efficiency of 3 x 10 bacteria/g of DNA, how many transformant colonies would be expected to grow on the LB/amp/ara plate? You can assume that the concentration of DNA and fraction of cells spread on the LB agar are the same as that of the pGLO laboratory.
3

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APPENDIX 1. FLUORESCENCE MICROSCOPY

When irradiated with short wavelength light, fluorescent substances emit light of a longer wavelength and non-fluorescent objects, such as the background, remain dark. In order to achieve the aim of rendering certain structures visible or of highlighting details for specific analysis, such specimens must first be stained or labelled with a fluorescent dye, known as a fluorochrome (e.g LIVE/DEAD Kit, Practical 3), or tagged with a fluorescent protein such as GFP (for example cells expressing GFP from the pGLO plasmid). The basic optical setup for epifluorescence microscopy and incident light excitation is below: The excitation filter is designed to have the highest possible transmission at those wavelengths which cause fluorescence in a particular specimen, while suppressing all remaining irradiation. The dichromatic mirror reflects the short wavelength excitation light onto the specimen, but is transparent to the longer wavelength fluorescence. The suppression filter absorbs the excitation light reflected from the specimen which re-enters the objective, but is highly transparent to the fluorescence wavelengths specific to the specimen. The interaction of the three filter components results in a bright, contrasty fluorescence image against a dark specimen background.

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APPENDIX 2: Leica Microscope Computer Protocol

Leica DFC camera connects to computer via Firewire (USB 2.0) cable and socket on the back of the camera and computer. Student logs onto class computer with Leica microscope and DFC camera attached. Go to Start > All Programs > Graphical > Corel DRAW Graphics Suite X5 > Corel PHOTO-PAINT X5 Close Quick Start window. Go to File > Acquire Image > Select Source Ensure Leica DFC Camera 7.3 (32-32) is selected, then click Select Then File > Acquire Image > Acquire Now, manually focus image on Leica microscope. If error message No camera connected is present in bottom left hand of DFC 280 window, or no source is present in Select TWAIN source window. Try unplugging the cable at both ends, then plugging back in again. Then try changing the cable, and finally, move the microscope to another computer. If none of these steps solves the problem- the camera may be the issue. Note: The drivers should be loaded onto all the computers in all labs to allow the microscopes to be used anywhere they are needed. If they are not apparent on the computer, or there are no TWAIN sources to select from- the drivers can be found at: Computer > APPS (S:) > Biological Sciences > leicadfc280 Then open: DFCTwainSetup_ENU.exe Use the admin login and password to access the file and follow the installation instructions.

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