From: Tsz Wong <twong103@[Link].
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To: Evelyn Holmes <eholmes3@[Link]>
Sent: Wednesday, March 4, 2015
Subject: Proposal for topic: Evaluation of antimicrobial activity against
pathogens using Lactic Acid Bacteria isolated from cheese and
yogurt
Hello Evelyn,
Please review my proposal and let me know if you need any additional information.
Summary
The proposed study will evaluate the potential of Lactic Acid Bacteria as a bio preservative. The
project entails isolating Lactic Acid Bacteria from yogurt and cheese and testing their
antimicrobial effectiveness again common foodborne pathogens. You approval of the topic is
requested so that I can begin the Formal Report.
Background
Lactic acid bacteria(LAB) has shown to be a promising biopreservative because they are
generally recognized as safe (GRAS) for human consumption and possess several intrinsic
properties that interests scientists (Chowdhury et al., 2012). Such properties includes the ability
to form organic acids and the ability to secrete of bacteriocin. Organic acids decrease the pH
environment, thus inhibiting the growth of many bacteria unable to tolerate high acidity, while
bacteriocins are antimicrobial compounds that kill a wide spectrum of bacteria including those
closely related (Teuber, 2009). The only bacteriocin ever to have become recognized as a legal
food preservative was Nisin (Teuber, 2009). Previous studies have shown Nisin’s bactericidal
action on gram-positive bacteria by forming pores in the cell membranes (Teuber, 2009). Thus, a
novel method is to use microorganisms such as LAB as a bioperservative to limit food spoilage
and food borne infections.
Proposal Plan
Lactic Acid Bacteria (LAB) will be isolated from yogurt and cheese using MRS agar and M17
agar. The ingredients in MRS media includes all the nutrients necessary for growth, furthermore
it contains ingredients that are slightly selective against organisms other than lactobacilli. M17
agar on the other hand, contains disodium-beta-glycerophosphate which inhibits growth of
Lactobacilli and allowing only Lactococcus to grow (Difco & BBL manual, 2012). In order to
assess the antimicrobial activity of lactic acid bacteria, the agar well diffusion method was used.
The principle behind the method is that a lawn of indicator bacteria is prepared, and the cell free
supernatant extracted from the LAB is inoculated into the wells of the agar. The cell free extract
diffuses out of the agar and if it possess antimicrobial properties, there should be appearance of
clear circular areas around the well where the bacteria is unable to grow. The size of the zones of
inhibition correlates to the effectiveness of the antimicrobial compounds against the respective
pathogen (Bonev et al., 2008).
In order to assess the antimicrobial activity of lactic acid bacteria, the agar well diffusion method
was used. The principle behind the method is that a lawn of indicator pathogenic bacteria is
prepared, and the cell free supernatant extracted from the LAB is inoculated into the wells of the
agar. The cell free extract diffuses out of the agar and if it possess antimicrobial properties, there
should be appearance of clear circular areas around the well where the pathogenic bacteria is
unable to grow. The size of the zones of inhibition correlates to the effectiveness of the
antimicrobial compounds against the respective pathogen (Bonev et al., 2008).
Project Schedule
Week 1 -3: Research on the topic and techniques used to perform the experiment
Week 4: Media preparation for Biochemical Tests used to confirm the identity of the
pathogens: [Link], Y. Enterocolitica, [Link]. Preparation of Biochemical Tests to
confirm Lactic Acid Bacteria.
Week 5: Media preparation for MRS and M17 agar used to isolate and grow Lactic Acid
Bacteria
Week 6: Isolation of Lactic Acid Bacteria from yogurt and cheese using serial dilution
and plating the samples onto MRS and M17 agar
Week 7: Perform Biochemical Tests and microscopy to confirm the identity of Lactic
Acid Bacteria from yogurt and cheese
Week 8: Preparation of the cell free supernatant containing antimicrobial compounds of
interest using Lactic Acid Bacteria that were isolated
Week 9: Preparation of pathogenic bacteria lawn and perform agar well diffusion test
Week 10: Analyze the effectiveness of the antimicrobial compounds by measuring the
zones of inhibition on the agar wells
Week 11: Repeat agar well diffusion test for reproducible Results
Week 12: Read results and compare with first trial of experiment
Reporting Schedule
Topic outline will be submitted on Week 4 1/28/2015
First draft evaluation will be submitted on Week 8 3/04/2015
Progress report will be submitted on Week 11 3/25/2015
Final report will be submitted on Week 13 4/08/2015
Action
The information above provides the reasons for the proposed study and outlines my tentative
work schedule. May I please have your approval by March 11 to use this topic for my formal
report?
References
Bonev et al. (2008) Principles of assessing bacterial susceptibility to antibiotics using
the agar diffusion method. J. Antimic. 61(6), 1295-1301
Chowdhury et al. (2012) Screening of Lactobacillus spp. from buffalo yogurt for probiotic
and antibacterial activity. J Bacteriol Parasitol. 3(8) 2155-9597
Difco & BBL Manual 2nd Edition (2012)
Teuber, M. (2009) Lactococcus. Bergey’s Manual of Systematic Bacteriology Vol.3.
Springer, New York. Pg. 711-719