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Introduction to Sterile Technique

Use sterile technique when pouring bacterial plates

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.


Needed Materials

155155 Micrococcus luteus, tube culture

826102 Nutrient Agar Slant

826122 Nutrient Broth, tube

821862 Nutrient Agar Prepared Media Plates

test tube rack

Bunsen burners and lighters

inoculating loops

beakers or other holders for inoculating loops

incubators

laboratory markers

culture tube labels

disinfectant (e.g., 70% ethanol, Lysol®, bleach solution)


Optional Materials

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).


Safety

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.


Procedures

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.


Optional

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™.


Answer Key to Questions Asked on the Student LabSheet

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

Retrieve your cultures and examine them, noting any changes.

Agar slant There is bacterial growth in the tube along the line of inoculation. The growth is yellow and matches the appearance of the growth seen in the original culture tube.

Broth The broth is cloudy, indicating bacterial growth.

Plate The surface of the agar is covered with bacterial growth, which is yellow and matches the appearance of the growth seen in the original culture tube. The growth follows the zigzag line of inoculation. Near the end of the streak, there may be individual colonies of bacteria.

Do you see any indication of contamination? If so, how might it have happened and how could you prevent it from reoccurring? There is no sign of contamination on the agar slant or in the plate culture. The growth matches the appearance of the original culture. I cannot tell from appearance whether the broth culture is contaminated or not. Note: If there is contamination, take note of the student’s ability to analyze the problem and suggest corrective actions.

Describe a simple procedure that you could use to determine whether or not your broth culture is contaminated. The intent of this question is to reveal something of the student’s background knowledge and reasoning skills. Some may describe isolation streaking or dilution methods. Others may suggest staining slides of the culture. Accept any answer that the student can support with a reasonable argument.





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