The replica-plating results are tabulated in Table 1. If aseptic technique is performed properly, cross-contamination i. The left panel shows a culture displaying uniform fine turbidity typical of a pure E. Wash floors and bench tops frequently with an appropriate disinfectant. Personal hygiene One common source for contaminating bacteria in a microbiology lab is the human beings carrying out the experiments. Always label all tubes and plates with: 1. Plastic tubes and tips cannot be flamed - these should be pre-sterilized by alternative methods prior to use.
. This reduces the chances for contamination and minimizes the consequences, if it does occur. If pouring must be done, remove any liquid from the threads with a sterile alcohol wipes or gauze pads. Instruments used for streak-plate technique. A needle can be used instead of a loop to inoculate an agar slant by stabbing the needle containing the inoculum into the agar Fig 1.
Do not place the cap on the laboratory bench but hold it between your ring finger and palm of the right hand while manipulating the pipette aid with the thumb, index and middle fingers of the same hand Figure 4. Using a serological pipette, first the broth must be aseptically transferred from the media bottle to the flask. Following incubation, secondary plates may be inspected and scored for growth versus no growth. Obligate aerobes are organisms that grow only in the presence of oxygen. It is a fundamental skill for working in a microbiology laboratory.
Consequently, many colonies grow along the outer rim of the plate. P2: For volumes between 0. Remove one pipette from the canister by holding it horizontally and gently shaking it so the tops of one or two pipettes stick out about an inch and can be easily grasped. After the disinfectant has dried completely, use an igniter to light the Bunsen burner. Lift the bottom half of an inverted plate from the bench then touch the loop, stick or toothpick to the first quadrant near the end of the last streak.
Four to five zigzags seems to work well. Allow the loop to cool a few seconds to avoid killing the inoculum. As shown, the strains show variable growth patterns following incubation on the secondary plates. Although both are turbid, the culture on the right has been contaminated with a fungus or other airborne microorganisms giving the culture a different color and consistency from that expected for E. At the Bench: A Laboratory Navigator.
The clearing is caused by hydrolytic enzymes secreted by the bacteria breaking down the cellulose in the medium. In this case, although the numbers are set identically on the volumeter, the wrong micropipettor was selected for the job the student used a P200 instead of a P20; panel B resulting in the delivery of a substantially larger volume of buffer. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. Replace the caps of flasks that have wet threads or wipe dry with sterile alcohol wipes. Since water has a density of 1, then 1 ml of water is equivalent to 1 gram g.
Streak the loop across the surface of the agar medium using the either the pattern shown in or the pattern shown in. For example, Serratia marcescens produces a deep red pigment at 25°C, but does not produce pigment at 37°C. This heats the glass and creates a convection current which forces air out of the tube and prevents airborne contaminants from entering the tube. Be careful not to gouge into the agar with the loop as you pick up the organisms Close the lid immediately once you have picked up the organisms and turn the plate upside down on the work surface. Further depressing the plunger to the second stop dispenses whatever liquid remains in the tip. Fig 1: Inoculation of culture into agar slant. Many research scientists prefer using disposable, pre-sterilized wooden sticks or flat toothpicks for streak-plating.
A pre-sterilized loop, stick or toothpick is used to spread the sample across one-quarter of the agar surface with a rapid, smooth, back-and-fourth motion from the rim to the center of the plate. Mesophiles are bacteria that grow best at moderate temperatures. If clean-up of the contaminated culture is attempted, then any work with this culture should be reserved to the very end of the day to minimize transfer of the contamination. For instance, it is critical for a wastewater treatment plant, which is responsible for cleaning up liquid waste e. Their optimum growth temperature is between 25°C and 45°C. For instance, those that adsorb during early exponential phase make larger plaques with more progeny phage than those that adsorb in late exponential phase. Discussion Aseptic technique refers to a set of routine procedures done to prevent sterile solutions and cultures from becoming contaminated by unwanted microorganisms in the laboratory.
B The left volumeter is from a P20 micropipettor, while the right volumeter is from a P200 micropipettor. Figure 8A provides an example of a pure versus contaminated culture of E. These streaking patterns allow you to obtain single isolated bacterial colonies originating from a single bacterium or arrangement of bacteria. The most common technical errors that occur with the soft-agar overlay technique are pouring the melted soft-agar either when it is too hot or too cool. Retention of these cultures poses a serious threat of cross contamination to other cultures in the laboratory. Pouring also increases the possibility of aerosol formation Caputo, 1988. Precisely read the volume drawn into the pipette by aligning the meniscus formed on top of the liquid column to the graduation marks on the calibrated pipette Figure 5.