Laboratory Investigations in Microbiology
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The Microbiology Laboratory

Equipment

Spectrophotometer

A spectrophotometer is an instrument which passes a beam of light through a liquid and measures how much of this light is absorbed by the liquid. The absorbance of a liquid is useful to know in several instances. For liquid bacterial cultures (broths) the absorbance is directly proportional to the cloudiness of the broth and therefore is a measure of the number of bacteria in the broth. For pigments, the absorption spectrum can help in identifying the type of pigment and the wavelengths of light it absorbs best.

Different models of spectrophotometers vary in how they are calibrated, but in essence the instructions are basically the same:

1. Turn on the spectrophotometer and allow it to warm up. 
2. Select the wavelength to be used using the up/down arrows
3. Insert a "blank" - a cuvette containing only the sterile liquid/solvent. Close the lid. Press the "Cal" button.
4. Remove the "blank" and insert a cuvette with the liquid to be measured. Close the lid and record the absorbance value displayed.

Microscopes

Our laboratory is equipped with (new in 2007) Leica DM-E binocular light microscopes, fitted with 4x, 10x, 40x, and 100x objective lenses for magnification ranging from 40 - 1000-fold. These microscopes feature the following controls:

• Power switch/dimmer switch
• Stage controls: move slides on the stage 
• Coarse and fine focus knobs
• Condenser lens control knob: raises and lowers the sub-stage condenser
• Iris diaphragm: adjusts the amount of light passing through the condenser

Digital cameras for microscopes

Each lab station will have use of a DM300 digital microscope camera, allowing you to capture still images of what you are looking at. The camera works in association with the ScopePhoto software on your lab station computers.

1. Focus and center the image you wish to photograph
2. Remove one eyepiece of your microscope and insert the camera into the eyepiece tube.
3. Connect the camera to the computer via the USB cable provided
4. Start your ScopePhoto software
5. Click on the SD button and select the DM300 camera
6. Click on the TC button. A live image should now appear in a new window on your screen
7. Once you are satisfied with the image you see, click the CAPTURE button.
8. Close the capture window.
9. View the image file. You can now use the SAVE AS function to save the image to your pen drive. (I recommend saving it as a jpg file)

Water baths

Water baths are used to bring culture media and cultures to a specified temperature. The set point of each water bath can be as high as ~ 100°C. These water baths are useful for high-temperature incubations (e.g. growth experiments at temperatures of 55°C, 65°C etc. ) and for keeping agar media liquid before pouring them into Petri plates. 

Incubators

There are two large walk-in incubator in the rear of the autoclave/glasswash area (Hudson 220A), set to a temperature of 30°C (left) and 35°C (right). Thess temperatures are ideal for the culture of most laboratory strains of bacteria and will be used for most of our experiments. Other, smaller incubators in the side of the lab are used for other incubation temperatures as needed.

pH Meters

There are several pH meters used to measure and adjust the acid/base balance of solutions.

Autoclave

An autoclave is an instrument used to sterilize culture media, plastic lab supplies such as pipette tips, and to sterilize all lab supplies prior to cleaning and disposal at the end of an experiment. Autoclaves heat materials to a temperature of ~ 121°C at a pressure of 103 kPa. This temperature and pressure kills all microbes - viruses, bacteria, spores and fungi (except one strain of Archaea!). The autoclave is located in Hudson 222A.

Bunsen burners

Bunsen burners are widely used in Microbiology to sterilize, heat-fix, and help to maintain a relatively germ-free work zone at the lab bench. The gas valves in this room are in the "ON" position when the handle is parallel to the spout, and "OFF" if the handle is at 90 degrees (on either side) of the spout. Turn the handle on, then use either a striker or a grill lighter to ignite the gas. Adjust the flame so that there is a bright blue inner cone, a darker blue outer cone, and no yellow flame visible. Adjust the flame to a height of ~ 6". The area of highest temperature is just above the bright blue inner cone.

Bacti-cinerators

An alternative to using a Bunsen burner when sterilizing inoculating loops and needles, the Bacti-cinerator produces the high temperatures for sterilizing the metal wire in a hollow ceramic chamber. Allow the chamber to warm up for 10 minutes before using. Once it is warmed up, hold the loop or needle wire into the chamber for 5 seconds for complete sterilization. The Bacti-cinerator may be left on all day and avoids the hazard of an open flame in the laboratory.  We will use the bacti-cinerators for most of the experiments this semester.

Micro-Pipetters 

Micro-pipetters are precision instruments which allow the accurate pipetting of small volumes of liquid. They are fragile and expensive, so please handle these with great care! There are different sizes of micro-pipetters. The two sizes we will use in this lab are the P-1000 (pipets up to 1000 microliters = 1 ml) and the P-200 (pipets up to 200 microliters, or 0.2 ml). The P-1000 pipetters have a blue knob at the top and are inscribed with the range (1000 - 100). The P-200 pipetters have a yellow knob and are inscribed with the range (200 - 20). To use the pipetters:

• Press a pipette tip on the pipetter. For the P-1000, the tips will be blue or clear in color, for the P-200 yellow.
• Select the volume you wish to pipette by turning the dial on the handle until the desired volume is displayed. For the P-1000, the top number is either 0 or 1 (ml), the numbers below it (under the red line) being the 100's and 10's of microliters. For the P-200 the top number is the 100's. Therefore, if the diplay reads 100 for the P-1000, it is set to pieptte 1 ml. 
• Depress the button (plunger) at the top until you come to a stop. Notice that it is possible to push down more - this reserve push is only for pushing out any extra liquid at the end of the pipetting.
• Insert the pipette tip into the liquid you wish to pipette and gradually ease the plunger out to fill the pipette

Vortexers

A vortexer is used for mixing solutions and suspensions in test tubes. It is especially useful when you want to get the bacteria in a test tube to be evenly mixed in the tube. You use the vortexer by holding the test tube near the top, with one finger on the cap, and lightly pressing the bottom of the tube into the cup of the vortexer until it starts. Make sure you are holding on tight to the test tube!

Magnetic Stir-plates and Hotplates

Hotplates are used in the laboratory for heating solutions. This is important in Microbiology when you make culture media because agar needs to be heated to ~80C before it will dissolve. (Note: Broths do not need to be heated) Heating may also be used to try to pasteurize or sterilize liquids. Many hotplates have a built-in magnetic stirrer that will keep your liquid stirring (using a magnetic stir-bar that is placed into your beaker or flask). 

© 2003 - 2017 José de Ondarza, Ph.D.