CAROLINA LABSHEETS
In this lab, students practice transfer of a bacterial culture using sterile technique. The lab is intended as an introductory microbiology activity. Before conducting the lab, ensure that students are familiar with standard lab procedures, including the use of gas burners and personal protective equipment.
Allow your students to practice important sterile lab skills for studying microorganisms.
Micrococcus luteus is recommended for this lab because it produces pigmented colonies that are visually obvious and is a biosafety level 1, nonpathogenic bacterium. M. luteus requires 2 to 4 days of incubation to get a good subculture for the second part of the lab. If this does not fit your schedule, consider 155452 Serratia marcescens D1, which is also pigmented and biosafety level 1 but may be ready 24 hours after subculturing. Do not use any bacterium for this lab that we list as a pathogen. If you have an autoclave, you can prepare your own agar slants, broth tubes, and plates using 785300 Nutrient Agar and 785360 Nutrient Broth. You may substitute tryptic soy agar for nutrient agar if desired (827302 TSA Agar Slant, 827372 TSA Broth Tube, 822022 TSA Plate, 788420 TSA Dehydrated Medium, and 788441 Tryptic Soy Broth Dehydrated Medium). Many labs are replacing traditional gas burners with infrared incinerators (703400 Carolina™ infrared Bacterial Sterilizer).
This activity requires that students work with bacterial cultures and open flames. Ensure that students use sterile technique at all times and wear appropriate safety equipment. All work surfaces must be wiped down with disinfectant before and after the lab. Have all students wash their hands before and after the lab. Destroy cultures remaining at the end of the lab by autoclaving or by flooding with disinfectant overnight before proper disposal. Review Techniques for Studying Bacteria & Fungi for more indepth information.
Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Use personal protective equipment such as safety glasses or goggles, gloves, and aprons when appropriate. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.
Have students work individually.
Have incubators on and operating at 25°C before the initial lab period. If cultures must be incubated at room temperature, this will lengthen the incubation time.
Materials (Micrococcus luteus cultures, nutrient agar slants, culture tubes of nutrient broth, nutrient agar plates, test tube racks, beakers, and inoculating loops) should be set out for student pickup. Alternatively, set up each student workspace with a test tube rack containing tubes of M. luteus culture, agar slants, and broth. At each workspace, have a burner and lighter.
The transfer to broth should be done first as that section of the student sheet contains the most complete instructions for flaming the loop and other basic procedures.
doing a demonstration or showing a video of the tube-to-tube inoculation technique used in the section “Transferring to an Agar Slant.” If holding both tubes for a transfer is too advanced for a student, they can revert to the one tube-at-a-time technique used in the “Transferring to Broth” section; however, if they will be continuing in microbiology, they should master the two tube technique.
All student cultures should be incubated at 25°C and examined at 24, 36, 48, 72, and 96 hours as needed. The second part of the lab can be done when there is obvious growth on the plates and in the tubes. This will usually be between 48 and 96 hours of incubation for M. luteus. Growth of M. luteus in broth usually lags about 24 hours behind its growth on agar.
If the original cultures are maintained at room temperature, students will be able to compare the originals and the subcultures.
Students can stain and view slides of their cultures. For directions, see our Introduction to Prokaryotes: Bacteria LabSheet. Students can streak their broth culture onto agar to check for purity of the culture. See our Isolating Bacteria from a Mixture LabSheet for instructions. The activity can be made more exacting by having them transfer two different bacteria onto media. If the students are not properly sterilizing their loops between transfers, they will be more likely to get contaminated cultures. You may want your students to seal their plates with tape. If so, we recommend 199278A or 199278D Petri-Seal™.
Note: The sample answers given below are for M. luteus. If you use a different bacterium, answers dealing with the appearance of the cultures will be different
*Next Generation Science Standards® is a registered trademark of Achieve. Neither Achieve nor the lead states and partners that developed the Next Generation Science Standards were involved in the production of this product, and do not endorse it.
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