enough material for a class of 30 students

Question: What does enough material for a class of 30 students mean?

Answer: For microbiology items(algae, protozoa, bacteria, fungi) this denotes that with the material provided, up to 30 students will be able to make a wet mount slide and examine relevant morphology for that organism.

Care guide

Caution
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This care sheet provides general information only for handling Carolina™ bacterial cultures. When you work with bacteria, it is imperative that you use sterile techniques at all times. Failing to use sterile techniques can contaminate cultures and work areas, and cause health and safety risks. See our Carolina™ Techniques for Studying Bacteria and Fungi Manual (item #154664) for descriptions of sterile techniques and standard practices for handling bacterial cultures.
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Immediate care and handling
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When your bacterial cultures arrive, immediately open the shipping container and remove the cultures. We ship cultures in tubes, plates, and as MicroKwik Culture® freeze-dried cells. Visually inspect each culture. Ensure that tubes are intact with caps securely in place, plates have no cracks and lids are secured by tape, and plastic bags containing MicroKwik Culture® cells are securely closed with the enclosed tube and vial intact.

You must seal any culture damaged during shipment—and anything it contaminated—in an autoclavable bag, and then sterilize it by autoclaving or soaking all contaminated materials in disinfectant. Note: Never discard a damaged, unsterilized culture in the trash. Know and follow your school or district’s guidelines for proper disposal. Contact our Customer Service at 800.334.5551 for a replacement of your damaged culture. It will help to have your order number available when you call.

Hold most cultures at room temperature, 20 to 22° C (68 to 73° F). For tube cultures, loosen the cap and keep the culture tube upright in a test tube rack or beaker. For best results, use cultures within 3 to 5 days of receipt; however, most bacterial cultures remain usable for up to 4 weeks when held at room temperature. Keep in mind that Carolina provides a variety of bacterial cultures and some have very specific care and handling needs that differ from these general guidelines. Vibrio fischeri, for example, must be kept in the dark and subcultured 2 to 3 times per week to maintain bioluminescence.

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Maintaining and culturing
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Eventually your bacterial culture will deplete the nutrients of the medium in or on which it is growing and will need subculturing. Before working with bacterial cultures, wash your hands with soap and water, ensure that the work area is draft free, and wipe the work surface with 70% alcohol or similar disinfectant. Note: Always check the culture for signs of contamination immediately prior to using. Never work in an area where food is prepared or consumed.

Transfer broth cultures to fresh broth using a sterile pipet or loop or streak onto agar using a sterile inoculating loop. For faster growth, you can incubate most cultures at 25 to 30° C (77 to 86° F). After making the transfers, clean the work area with disinfectant and wash your hands again. Either autoclave the old stock cultures and glassware, or cover them with disinfectant overnight. We recommend the use of Clavies® Autoclavable Bags (item #831642) for disposal.

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Biosafety
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In the US the CDC (Centers for Disease Control) sets standards for the safe handling of microorganisms according to their Biosafety Level (BSL). There are 4 Biosafety Levels with BSL-1 being the lowest risk microorganisms and BSL-4 being the highest risk. Bacterial cultures provided by Carolina Biological Supply Company fall into BSL-1 and BSL-2. Our catalog and online listings identify BSL-2 bacterial cultures as pathogens. They are only available as MicroKwik Culture® freeze-dried cells, and we can only ship them to colleges and universities. Cultures not identified in our listings as pathogens are BSL-1.

The CDC standards for working with BSL-1 microorganisms include the following:
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Follow all standard microbiological practices.
Work can be performed on an open lab bench or table.
Wear personal protective equipment (PPE), i.e., lab coats, gloves, and eye protection, as needed.
A sink for hand washing.
Doors that separate the lab room from the rest of the facility.

For working with BLS-2 microorganisms the CDC recommends all of the above, plus the following:
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Restricted access to the lab room while cultures are being used.
PPE to include face shields as needed.
All procedures that can produce a splash or an aerosol must be performed within a biological safety cabinet.
An autoclave or other method of decontamination for proper disposal.
Self-closing doors that separate the lab room from the rest of the facility.
An eyewash station.

For a fuller discussion of CDC criteria for handling microorganisms see the following link: http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf [link].

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FAQs
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Which agar should I use for my bacterial cultures?
In Carolina’s print and online catalogs, the product descriptions for our bacterial cultures include the type of nutrient medium that we use for each culture. This information also appears on the label affixed to the culture. A wide range of bacteria will grow on Nutrient Agar (item #821862) and Tryptic Soy Agar (item #822022).

Can I pour my own plates if I don’t have an autoclave?
See our Nutrient Agar Bottle (item #776360) and other prepared media bottles. A bottle containing 125 mL of medium will pour 4 to 5 standard 100 × 15-mm plates. Watch our “How to Melt and Pour Agar Plates” video (at www.carolina.com) for a demonstration of the technique.
Should I order a tube, plate, or MicroKwik Culture®?
Tube cultures are best for stock. A tube culture is often streaked on a plate and incubated before lab use. Order a plate culture for convenience and immediate use. You can receive a plate culture and use it in a lab on the same day. A MicroKwik Culture® is best used for longer-term storage and to receive a culture of a pathogen. A MicroKwik Culture® can be held at room temperature for up to 2 months before being activated or for 6 to 8 months refrigerated.

Which cultures should I order for antibiotic testing?
Gram-positive and Gram-negative bacteria often give different results when tested against the same antibiotic. For that reason, we recommend testing against Bacillus cereus (item #154872), which is Gram-positive, and Escherichia coli (item #155068), which is Gram-negative. We recommend broth cultures because this makes it easier to spread the culture over the surface of an agar plate. See our Carolina BioKits®: Antibiotic Sensitivity kit (item #154740) for more information.
You recommend holding a culture at room temperature, but the recommended temperature for Escherichia coli (item #155065) is given in your catalog as 37° C. Why is that?
Thirty-seven degrees C is the incubation temperature, the temperature needed for maximum culture growth. The culture is mature when we ship it out and does not need further incubation. Maintaining the culture at room temperature allows you to hold it longer before use.

Care guide

Immediate care and handling

When your protozoa culture arrives, immediately open the shipping container, remove the culture jars, and inspect them for any signs of damage. Once you have verified that the shipment is intact and not damaged, loosen the lids on the jars. Aerate the cultures using the plastic pipet supplied with each individual culture. Use a different pipet for each culture, and write the name of the culture on each to avoid contamination. Aerating helps replace the oxygen depleted during shipment. To aerate, place the top of a pipet into the culture water and squeeze the bulb, bubbling air into the water. Withdraw the pipet and release the bulb, allowing it to refill with air. Repeat the above process 4 more times.

Sampling and observation

Allow 15 to 20 minutes after aeration for the organisms to settle. Inspect the culture using a stereomicroscope and low illumination. Using the stereoscope will allow you pinpoint the areas where the organisms are concentrated. In preparing slides for viewing, students should obtain their samples from those areas. Using the stereoscope and designated pipet for that culture, students can pick out a single organism (or group of organisms) for their individual slides.

Many protozoans (such as Paramecia) concentrate in areas where food particles are abundant. These areas are visible as fuzzy debris within the culture. Amoeba can be difficult to locate at first because they move slowly and lack a constant shape. To find them, focus on the bottom of the jar after it has been sitting undisturbed for at least 15 minutes. Watch through the stereomicroscope for a few seconds and you should begin to see dozens of Amoeba as they creep slowly across the bottom. You will also see a faster moving organism called Chilomonas (a tiny flagellate) moving about the Amoeba culture. Chilomonas serves as a food source for the Amoeba. Stentor tends to attach to the sides of the culture jar. Other varieties may concentrate near the surface of the water.

To pick up an organism (or organisms), squeeze the pipet bulb before inserting the pipet into the culture. Release the bulb when the pipet’s tip is close to the concentration of protozoans. Keep the pipet vertical as you are drawing the sample to avoid stirring up the culture and scattering the organisms. Do not squirt the pipet water back into the culture.

One drop should contain more than enough organisms for a slide mount. After adding a coverslip, examine the slide using the microscope’s lowest magnification. After protozoans have been located, high magnification can be used to observe the organisms in greater detail. Some protozoans, mainly ciliates, move so rapidly that it can be difficult to keep them in the field of view under high-power magnification. A quieting solution, such as Protoslo^® (item #885141), slows the ciliates’ movement without killing them. Add 1 drop of Protoslo^® to a drop of culture on a slide, mix well, add a coverslip, and observe.

Care and culturing

Photosynthetic protozoans (Euglena and Volvox) need light to manufacture their own food. Use either indirect natural light or a light bank (such as our item #158999) to provide light for these organisms.

Never place protozoa cultures in a refrigerator or in direct sunlight. Maintain cultures between 20 and 22° C (68 and 72° F), with the lid placed lightly over the mouth of the jar. Plan to use the culture as soon after receipt as possible.

Many protozoans are easily cultured. For further instructions on how to culture protozoa, refer to the Carolina^™ Protozoa and Invertebrates Manual.

FAQs

How long can I keep my cultures before using them?

If possible, use them within 2 to 3 days of receipt; however, most protozoan cultures will remain useable for a week or longer.

Will the cultures last longer if kept in the refrigerator?

We do not recommend refrigeration or rapid temperature changes of any kind for our cultures.

Are these protozoans dangerous?

No, the protozoans in our collection are for general classroom use. They are not parasitic or pathogenic in any way.

My cultures arrived on a Friday. Can I keep them over the weekend and use them for class on Monday?

Remove the cultures from their shipping container and care for them as directed in the “Immediate care and handling” section. Do not leave them in the unopened shipping container. Photosynthetic forms need light. Heterotrophic forms are shipped with enough food in their jar to maintain the culture for an extended period of time. You may even find that the cultures improve when left over the weekend because they have had time to recover from the shipping process.

My students are not finding any protozoans. What should I do?

The culture may need to be agitated to redistribute the protozoans. Ensure students are following the sampling procedures described in the “Sampling and observation” section. Protozoan species differ greatly in size. If students have previously observed larger protozoa, such as Paramecium, remind them to look for smaller organisms when searching for Euglena. Finally, examine the culture under a stereomicroscope to locate areas where protozoans are concentrated and direct students to collect samples from those areas.

We used Protoslo^®, but now the protozoans are all at the edge of the coverslip and some have even been squeezed out completely from under the coverslip. What can I do?

If the Protoslo^® and culture water are not thoroughly mixed, the thicker Protoslo^® will displace the water and protozoans when the coverslip is added. If this occurred, clean the slide and start over, being sure to thoroughly mix the Protoslo^® and culture water before adding the coverslip to the slide.

Problems?

We hope not, but if so, contact us. We want you to have a good experience.

Orders and replacements: 800.334.5551, then select Customer Service.

Technical support and questions: caresheets@carolina.com