Lab Experiment III. Effect of Bee Venom on Microorganisms

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Denise Esqueda, David Hawa


Effect of Bee Venom on Microorganisms



Hypothesis:

If bee venom is introduced into a living sample of microorganisms under ideal growth conditions it will act as a colonial growth inhibitor. As concentrations of bee venom are increased it will adversely affect colonial growth of the specific microorganism.


Purpose:

Main observation is to compare Bee venom (BV) when autoclaved and not autoclaved, to see if denaturation of proteins in BV may result on full growth or no growth in 2 different procedures:# Twofold dilution: having diluted the BV in DI water in container, follow procedure on 1µg/1mL. DI H2O, on total of 7 cuvettes of 0, ½, 1/4, 1/8, 1/16, 1/32 and a negative control (DI H20).

  1. Kirby-bauer: Sixteen petri dishes will be used to compare the zones of growth inhibition in both autoclaved and non-autoclaved BV samples at varying concentration levels. Each plate will contain TSA and will be inoculated with both microorganisms and bisected through the center. After inoculation 20µL of the designated BV sample from the serial dilution will be applied to a sterile paper disc. After BV sample application the disc will be placed near the middle it’s bisected zone on the agar medium in the petri dish. This same procedure will be followed for both microorganisms and at all concentration levels. Twenty microliters of distilled water will be applied to a sterile disc and act as a negative control in this experiement.



Requested Microorganisms:

To be determined – Gram Positive, Bacillus, Endo spores (broth)

Klebsiella pneumoniae – Gram Negative, Bacillus (broth)


Materials:

*Bee venom (BV) is in powdered form and will be mixed with DI water at the following ratio:

1µg Bee Venom/1mL


24 cuvettes (serial dilution) Micropipetter (200µL-500µL)
16 petri dishes (bisected) Micropipetter tips
Paper discs (autoclaved) Inoculation Loops
spectrophotometer Metric measuring device/calipers
Wax pencil

*Bee Venom is to be provided by students








Procedures:

The step-by-step procedural testing processes will follow the guidelines as detailed in the Laboratory Exercises in Microbiology Tenth Edition by John P. Harley.


Procedure Name Function/Characteristic Page Number
(a) Gram Stain Determination Gram +/- 54
(b) Schaeffer-Fulton Procedure Endospore Staining 66
(c) Kirby-Bauer test Measurement/comparison of zones of growth.



Microorganism: Klebsiella pneumoniae
Autoclaved Sample Crude Sample
Concentration Zone of Inhibition in mm
Concentration Zone of Inhibition in mm
Negative

Control: DI Water

Negative

Control: DI Water

1µg/mL
1µg/mL
1/2
1/2
1/4
1/4
1/8
1/8
1/16
1/16
1/32
1/32
Microorganism: To Be Determined
Autoclaved Sample Crude Sample
Concentration Zone of Inhibition in mm
Concentration Zone of Inhibition in mm
Negative

Control: DI Water

Negative

Control: DI Water

1µg/mL
1µg/mL
1/2
1/2
1/4
1/4
1/8
1/8
1/16
1/16
1/32
1/32


Serial Dilution Schematic with Bee Venom and TSB


Original Sample BV in DI H20








1µg/mL


1µg/mL

2mL BV

1/2

1mL TSB/ 1mL BV

1/4

1mL TSB/ 1mL BV

1/8

1mL TSB/ 1mL BV

1/16

1mL TSB/ 1mL BV

1/32

1mL TSB/ 1mL BV

Articles:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043086/


[../../../E:/Microbiology/J.%20Biol.%20Chem.-1982-Terwilliger-6016-22-Bee%20venom%20melittin%20article.pdf file:///E:/Microbiology/J.%20Biol.%20Chem.-1982-Terwilliger-6016-22-Bee%20venom%20melittin%20article.pdf]
https://www.drugs.com/pro/wasp-and-bee-venom.html
https://www.medicines.org.uk/emc/medicine/19596