Jun 23, 2026
Protocol for Preparing Bacterial Glycerol Stocks from Honey Bee Brood Caps
- Cristina L Andujar-Sierra1,
- abigail.strubbe 1,
- Lizbeth Alvarado-Vargas1,
- Ariana Rodríguez-Flores1,
- miguel.urdaneta 1,
- Yilmaz Koru1,
- Cid M Calderon-Rodriguez1,
- Jose Agosto-Rivera1
- 1Universidad de Puerto Rico, RIo Piedras
Protocol Citation: Cristina L Andujar-Sierra, abigail.strubbe , Lizbeth Alvarado-Vargas, Ariana Rodríguez-Flores, miguel.urdaneta , Yilmaz Koru, Cid M Calderon-Rodriguez, Jose Agosto-Rivera 2026. Protocol for Preparing Bacterial Glycerol Stocks from Honey Bee Brood Caps. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5w1odv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2026
Last Modified: June 23, 2026
Protocol Integer ID: 319477
Keywords: bacterial glycerol stocks from honey bee brood cap, preparing bacterial glycerol stock, bacterial glycerol stock, honey bee brood cap, set of pure bacterial culture, material for bacterial identification experiment, pure bacterial culture, bacterial colony, bacterial identification experiment, glycerol
Funders Acknowledgements:
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321760
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321761
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321759
USDA-OPPE (NIFA)
Grant ID: AO242501X443G006
Abstract
This protocol explains how to prepare bacterial glycerol stocks from honey bee brood caps. Samples are processed, plated, and used to isolate bacterial colonies that can be stored for future lab work.
The expected result is a set of pure bacterial cultures preserved at −80 °C in glycerol. These stocks can be used as starting material for bacterial identification experiments, such as 16S rRNA analysis.
Materials
| Material | Supplier / Catalog | Quantity | |
| Microcentrifuge tubes, 1.7 mL (new) | VWR, Cat. No. 87003-294 | 25 | |
| Bistoury / small metal spatula | Any supplier | 1 (sterilizable) | |
| Inoculation loop | Any supplier | 1 | |
| Ethyl alcohol 70% | Any supplier | 100 mL | |
| Matches or lighter | Any supplier | 1 | |
| Permanent marker (Sharpie) | Any supplier | 1 | |
| Analytical balance | Any supplier | 1 |
Sample Collection Materials
| A | B | C | |
| Tryptic Soy Broth (TSB) powder | VWR, Cat. No. 90000-344 | 2.4 g (for 80 mL) | |
| Tryptic Soy Agar (TSA) powder | VWR, Cat. No. 90000-050 | 24 g (for 600 mL) | |
| Distilled water | Any supplier | 700 mL total | |
| Erlenmeyer flask, 500 mL | Any supplier | 1 (TSB) | |
| Erlenmeyer flask, 1000 mL | Any supplier | 1 (TSA) | |
| Culture tubes with caps | Any supplier | 25 | |
| Petri dishes | VWR, Cat. No. 25384-342 | 25 | |
| Glass beads (for spreading) | Any supplier | Per plate | |
| Micropipette (2–20 µL, 100–1000 µL) | Any supplier | 1 of each | |
| Sterile micropipette tips | Any supplier | As needed | |
| Hot plate with magnetic stirrer | Any supplier | 1 | |
| Magnetic stir bar | Any supplier | 1 | |
| Autoclave tape | Any supplier | As needed | |
| Polytron PT 1200 CL saw-tooth homogenizer | Any supplier | 1 |
Culture Media and Dilutions Materials
| Culture Media and Dilutions | Culture Media and Dilutions | Culture Media and Dilutions | |
| Culture Media and Dilutions | Culture Media and Dilutions | Culture Media and Dilutions | |
| Culture Media and Dilutions | Culture Media and Dilutions | Culture Media and Dilutions | |
| Culture Media and Dilutions | Culture Media and Dilutions | Culture Media and Dilutions | |
| Culture Media and Dilutions | Culture Media and Dilutions | Culture Media and Dilutions |
Glycerol Stock Materials
Safety warnings
- Flame sterilization: Bistoury, spatula, and loops are sterilized with ethanol and open flame. Allow instruments to cool completely before contacting biological material. Never wave a flaming instrument near alcohol bottles.
- Autoclave: Bistoury, spatula, loops and culture media must be autoclaved before use. Follow institutional autoclave operating procedures. Do not seal culture media containers completely before autoclaving; pressure buildup may cause containers to rupture.
- Hot plate: Media preparation involves heat. Use heat-resistant gloves when handling hot flasks. Risk of burns and boiling over if not monitored continuously.
- Hot agar: Molten TSA is very hot after autoclaving. Allow to cool to approximately 50–55 °C before pouring (Zimbro et al., 2009).
- Glycerol stocks: -80 °C freezer requires proper protective use gloves. Do not allow the glycerol stock to thaw; freeze-thaw cycles reduce viability.
- Autoclave caution: Do not seal the bottle completely before autoclaving. Pressure buildup may cause the container to rupture.
- Instrument caution: Never flame the instrument near an ethanol bottle. Allow the instrument to cool before touching biological material to avoid killing surface bacteria.
- Pipette caution: Change pipette tips between every transfer. Reusing tips carries over concentrated bacteria and invalidates the dilution series.
- Glycerol stock caution: Do not freeze-thaw the glycerol stock repeatedly. Each freeze-thaw cycle reduces bacterial viability. To subculture, scrape frozen material from the well surface with a sterile loop or toothpick without thawing the plate.
Before start
Complete all steps below before beginning sample collection.
- Prepare and autoclave all culture media (TSB and TSA) at least one day before sample collection. See Section 5, Phase 1 for detailed media preparation instructions.
- Pre-weigh all 1.7 mL microcentrifuge tubes to be used for brood cap collection. Record each tube's weight before going to the field.
- Pre-label all tubes with hive number and collection date before going to the field.
- Sterilize bistoury, spatula, and forceps with 70% ethanol and flame. Allow to cool before use.
- Prepare 3 culture tubes with 9 mL of TSB per hive (15 total for 5 hives) for serial dilutions.
- Prepare Petri dishes with TSA: pour 20 mL of cooled, molten TSA per plate. Allow to solidify, invert, and store at 4 °C until use. Prepare a minimum of 8 plates per hive (40 total for 5 hives).
- Verify that the homogenizer, hot plate, magnetic stirrer, micropipettes, and autoclave are available and functional.
- Prepare the 96-well plate layout for glycerol stock archiving before the plating step. Assign a well address to each isolate.
Culture Media Preparation: Tryptic Soy Broth (TSB)
Weigh 2.4 g of TSB powder on a weighing boat and transfer into a 500 mL Erlenmeyer flask. Add 80 mL of distilled water and stir manually until partially dissolved. Add a magnetic stir bar, place on a hot plate, and apply medium heat with continuous stirring until the solution becomes fully clear and translucent. Do not allow the solution to boil. (0.03 g/mL × 80 mL = 2.4 g; scale proportionally for other volumes.)
Distribute the dissolved TSB into sterilized culture tubes according to the volumes below.
| Tubes | Volume per tube | Purpose | |
| 1 | 5 mL | Homogenization | |
| 15 (3 per hive × 5 hives) | 9 mL | Serial dilutions (T1, T2, T3) | |
| 20 | 2 mL | Glycerol stock preparation |
Cap each tube loosely, apply autoclave tape, and autoclave according to institutional protocol (typically 121 °C, 15 min, 15 psi). Store in a sterile refrigerator until use.
Culture Media Preparation: Tryptic Soy Agar (TSA)
Weigh 24 g of TSA powder on a weighing boat and transfer into a 1000 mL Erlenmeyer flask. Add 600 mL of distilled water and stir manually until partially dissolved. Add a magnetic stir bar, place on a hot plate, and apply medium heat with continuous stirring until the solution becomes fully clear and translucent. Do not allow the solution to boil. (0.04 g/mL × 600 mL = 24 g; scale proportionally for other volumes.)
Transfer the dissolved agar to a glass bottle. Cap loosely , do not seal completely and apply autoclave tape. Autoclave according to institutional protocol (typically 121 °C, 15 min, 15 psi). Do not seal the bottle completely before autoclaving pressure buildup may cause the container to rupture.
After autoclaving, allow to cool to approximately 50–55 °C (warm to the touch, not hot). Pour 20 mL into each Petri dish in a sterile environment. Do not pour above 65 °C (condensation on lids) or below 50 °C (agar begins to solidify in the bottle).
Allow plates to solidify at room temperature, then invert and store at 4 °C until use. Each 600 mL batch yields approximately 28–30 plates. For 5 hives (130 plates total), prepare 4–5 batches.
Brood cap collection and homogenization: 20 brood caps per hive
Remove one frame containing sealed brood cells (capped pupae) from the selected hive. Open the sterile packaging of the bistoury and carefully excise 10 brood caps, placing them directly into the corresponding pre-labeled, pre-weighed 1.7 mL microcentrifuge tube. Throughout the collection, re-sterilize the bistoury frequently by dipping in 70% ethanol and passing through the flame. Allow to cool briefly before each contact with the brood caps.
CAUTION: Never flame the instrument near the ethanol bottle. A hot bistoury will kill surface bacteria and compromise the sample.
Cap the tube and transport to the laboratory on ice.
In the laboratory, weigh the tube containing the brood caps. Calculate the net brood cap mass: weight of tube with brood caps (mg) − weight of empty tube (mg). Add 1 mL of TSB per mg of brood cap mass directly to the collection tube. (Example: 50 mg brood caps → add 50 mL of TSB.)
Homogenize the sample for 3–4 minutes using the manual homogenizer (Polytron PT 1200 CL saw-tooth homogenizer) until the material is uniformly dispersed. The resulting suspension is the primary homogenate. Proceed immediately to serial dilutions.
CAUTION: When processing samples from multiple hives, the homogenizer probe must be cleaned and sterilized between hive samples to prevent cross-contamination. Remove visible residue, sterilize with 70% ethanol and flame, and allow the probe to cool completely before contacting the next sample.
Serial Dilutions
Label 3 culture tubes per hive (T1, T2, T3), each containing 4.5 mL of TSB. Transfer 1 mL from the primary homogenate into T1, mix by pipetting, and change tip. Transfer 0.5 mL from T1 into T2, mix, and change tip. Transfer 1 mL from T2 into T3, mix, and change tip.
Note: Change tips between every transfer, reusing tips invalidates the dilution series. Reference video (serial dilution technique:: https://www.youtube.com/watch?v=yYWFX4IXc5Y&t=22s)
| Tube | Dilution | Source | TSB volume | Volume transferred | |
| Primary homogenate | 1/1 (undiluted) | Brood caps + TSB | 1 mL/mg | — | |
| T1 | 1/10 | Primary homogenate | 4.5 mL | 0.5 mL | |
| T2 | 1/100 | T1 | 4.5 mL | 0.5 mL | |
| T3 | 1/1000 | T2 | 4.5 mL | 0.5 mL |
Figure 1. Serial dilution scheme for bacterial enumeration from brood cap homogenate. Three 1:10 serial dilutions are prepared (T1 = 1/10, T2 = 1/100, T3 = 1/1000) by transferring 0.5 mL into 4.5 mL of TSB. Two duplicate plates are plated per dilution. The original sample is also plated. The dilution producing 20–200 well-separated colonies is selected for monoculture isolation.
Plating and Incubation
For each hive, pipette 100 µL of each sample onto the center of a pre-labeled TSA plate. Add sterile glass beads, close the plate, and shake gently to spread the inoculum evenly. Discard the beads into a container with 70% ethanol after each plate do not reuse between samples without sterilization.
Label each plate with: dilution factor, hive number, date, and initials. Prepare the following plates per hive:
| Plate | Source | Number of plates | |
| Undiluted homogenate | Primary homogenate | 1 | |
| 1/10 dilution | T1 | 2 (duplicate) | |
| 1/100 dilution | T2 | 2 (duplicate) | |
| 1/1000 dilution | T3 | 2 (duplicate) |
Invert plates and incubate at 37 °C for 24–48 hours.
Colony selection and isolation
After incubation, photograph each plate using a colony-counting app to estimate CFU and document colony morphology. Select the plate with well-separated, non-overlapping colonies for each hive , this is the ideal plate for isolation.
Using an inoculation loop, pick up to 20 individual colonies per hive. Streak each onto a separate TSA plate using the streak plate technique to obtain pure isolates (progressively dilutes the inoculum across four quadrants so individual cells grow into isolated colonies (see Figure 2). Label each isolation plate with: colony number, hive number, dilution source, date, and initials.
Figure 2. Streak plate technique for bacterial colony isolation. The inoculum is progressively diluted across four quadrants by rotating the plate and streaking from the previous quadrant. Individual isolated colonies (shown in the bottom plate) arise in the final quadrant, each originating from a single cell and representing a pure monoculture.
Invert and incubate at 37 °C for 24–48 hours.
Next step: After incubation, confirm colony isolation. Proceed to: Glycerol Stock Preparation.
Glycerol Stock Preparation
Label the 96-well plate with the hive number, collection date, and plate identifier. Assign one well per bacterial isolate. Dispense 100 µL of TSB into each well to be used. Using a sterile inoculation loop or toothpick, transfer several colonies from each pure isolation plate into the corresponding well. Mix until the suspension is visibly turbid insufficient turbidity indicates too few cells and may result in poor recovery after freezing (Rawi et al., 2025).
In a separate 96-well plate, dispense 100 µL of 50% glycerol into each corresponding well. Transfer the 100 µL TSB + bacteria suspension into the matching glycerol well and mix gently by pipetting (final volume 200 µL = 25% glycerol final concentration; Rawi et al., 2025).
Seal the plate with adhesive film or a plate sealer. Label with hive number, date, and plate number. Place immediately at −80 °C, do not allow the plate to sit at room temperature. Avoid repeated freeze-thaw cycles, which reduce bacterial viability; subculture by scraping frozen material with a sterile loop without thawing the plate.
IMPORTANT: Glycerol stocks stored at −80 °C are stable for several years provided the freezer maintains a consistent temperature (Rawi et al., 2025). Temperature fluctuations significantly reduce viability.
Next step: Glycerol stocks are now ready for use. In the student laboratory session, a subculture from this stock will be plated on TSA and incubated for 24–48 h at 37 °C to obtain fresh colonies for cell lysis and PCR.
Protocol references
- Protocols.io. Preparation of bacteria glycerol stocks. https://www.protocols.io/view/preparation-of-bacteria-glycerol-stocks-b85iry4e.pdf
- Huemer, M., Mairpady Shambat, S., Brugger, S. D., & Zinkernagel, A. S. (2020). Antibiotic resistance and persistence — implications for human health and treatment perspectives. EMBO Reports, 21(12), e51034. https://www.sciencedirect.com/science/article/pii/S0167701208001632
- Glass bead spread plate technique [Video]. YouTube. https://youtu.be/ArWRREJl1Rw
- Glycerol stock preparation [Video]. YouTube. https://www.youtube.com/watch?v=Za8sJFpU8QU
- Nyre, L. M., Sundsfjord, A., Nymoen, D. Å., & Hanssen, A. M. (2015). Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLOS ONE, 10(2), e0117617. https://doi.org/10.1371/journal.pone.0117617
- BD Diagnostics. (2024a). Tryptic Soy Broth (TSB) — Product insert (cat. #90000-344). Becton, Dickinson and Company. https://us.vwr.com/store/catalog/product.jsp?catalog_number=90000-344
- BD Diagnostics. (2024b). Tryptic Soy Agar (TSA) — Product insert (cat. #90000-050). Becton, Dickinson and Company. https://us.vwr.com/store/catalog/product.jsp?catalog_number=90000-050
- Rashamuse, K., Sanyika, W., Mafokoane, M., & Makhalanyane, T. (2015). Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples. MethodsX, 2, 255–261. https://doi.org/10.1016/j.mex.2015.04.004
- Rawi, M. H., Zulkifly, S., Yaacob, N. H., & Sikin, A. M. (2025). Comparison of microbial preservation methods: a narrative review. Frontiers in Microbiology. https://pmc.ncbi.nlm.nih.gov/articles/PMC12013664/
Acknowledgements
This protocol was supported by the NSF Arecibo Center for STEM Education, Computing, and Community Engagement (Grant Nos. 2321760, 2321761, 2321759) and USDA-OPPE (NIFA) (Grant No. AO242501X443G006).