1
Formulation of a new specific
functional probiotic product
supplemented with nutraceuticals
AY 21- 22
School of
Life
Sciences
TABLE OF CONTENTS
1. PROJECT OVERVIEW———————————————————3
1.1 Background——————————————————————-3
1.2 How can you steer the project and make it your own?————————–3
1.3 What are the expectations for attendance?—————————————–3
1.4 The objectives of your project———————————————————-4
1.5 What outputs are expected?————————————————————4
2
1.6 What skills will you gain or develop in this project?——————————-4
1.7 Relation to current Food Microbiologist or analysists—————————–5
1.8 Health and safety considerations——————————————————5
1.9 Summary of expected lab work———————————————————6
1.10 Materials required————————————————————————-
9
2. FLOW DIAGRAM OF WORKFLOW——————————————————-10 3.
SUGGESTED PROTOCOLS—————————————————————-11
3.1 Enumeration- serial dilution————————————————————-11
3.2 Enumeration- Miles and Misra method———————————————–12
3.3 Isolation of Probiotics organisms——————————————————13
3.4 Streak plate method (pure culture)—————————————————-13
3.5 Phenotypic Identification: Colony morphology————————————–14
3.6 Gram staining and Microscopic observation—————————————-15
3.7 Simple biochemical identification and API CH test——————————–16
3.8 Genotypic Identificaiton: Genomic DNA extraction and 16S rRNA gene
amplification———————————————————————————17
3.9 Setting up 16S rRNA PCR reaction—————————————————18
3.10 Agarose gel electrophoresis of DNA fragments—————————————–18
3.11 PCR clean up————————————————————————————20
3.12 Sample preparation for sequencing 16S rRNA gene————————-21
3.13 Bacterial growth Assays————————————————————–23
3.14 Bacterial growth Synergistic and antagonistic plate assay——————24
3.15 Probiotics formulation and Survival method (Independent method)——25
4. RESOURCE————————————————————————————–25
1. PROJECT OVERVIEW
1.1 Background
Probiotics are live microbial communities used as food additives to exert various health
benefits to humans. Lactic acid Bacteria (LAB) are the centre point of probiotics. The
predominant probiotic bacteria are Lactobacillus, Lactococcus, Bifidobacterium,
3
Pedicoccus, Streptococcus, Enterococcus, Leuconostoc and Saccharomyces
boulardii (Kechagia et al., 2013; Karami et al., 2017). Various studies have claimed
that probiotics and their metabolites maintain a healthy gut by preventing pathogen
entry, boosting the immune system, and exhibiting anti-diabetic, anti-obesity, and
antiinflammatory properties (Kerry et al., 2018; Bermudez-Brito et al., 2012). One of
the valuable metabolites produced by gut microbiota is Short-chain Fatty acids
(SCFAs), such as acetate, butyrate, propionate. The SCFAs modulate colonic
physiology and motility and play a significant role in the gut-brain axis (Rastelli et al.,
2018). Several probiotic products are available in the market, and the demand for
probiotics is very high.
The current project aims to develop a new probiotic formulation with nutraceuticals.
Students will choose two products of their interest to isolate and identify probiotic
strains and investigate their labelling claims. In addition, students design and develop
a new probiotic product with supplemented beneficial nutrients or nutraceutical
ingredients, e.g. mushroom extract, milk or soy juice and prebiotics; analyse the
nutrient changes, and consider the specific custom group, e.g. diabetes. Furthermore,
students investigate some of the nutrient ingredients linked to their bioactive efficacy.
1.2 How can you steer the project and make it your own? You will isolate and
identify probiotic bacteria from commercial probiotic drinks/food. Each student must
choose at least two different probiotics bacteria and generate your own research
question and hypothesis to make the project distinct.
1.3 What are the expectations for attendance? Laboratory work will be completed
over the course of four weeks. During this time, you will be required to attend three
days per week (Mondays, Wednesdays and Fridays). You will be performing
experiments that have never before been carried out. Data is therefore not available
for those who are unable to attend in person, so, attendance is compulsory.
1.4 The objectives of your project: 1) quantify probiotics from the commercial
probiotic product using standard serial dilution and plating and Miles and Mishra
method 2) isolate a pure culture of probiotics using selective media MRS (De Man,
Rogosa and Sharpe broth/agar) media 3) identify the probiotic strains and investigate
4
their labelling claims using phenotypic and genotypic characteristics 4) perform
competitive growth assays to investigate the synergistic and antagonistic behaviour of
the probiotics 4) formulate a new probiotic drink with added value supplement.
1.5 What outputs are expected? Project report: You will critically review the literature
on Probiotics and your probiotic bacteria of choice. You will propose a research
question and hypothesis and report the results of your experiments. You will
statistically analyse your results where appropriate. Finally, you will present your
results as figures and describe their meaning in words.
• Bacterial enumeration data from the commercial probiotic food
• Pure culture isolation data
• Identification of bacteria- biochemical identification, 16S rRNA data
• Bacterial growth assay: monoculture, and combination of probiotics strain and
also with the nutraceuticals
You will discuss your results on probiotics research data by comparing them to the
probiotics published literature. Reflective Record: You will write daily entries in a
Research Record (lab book) that records all aspects of your research project, including
your research, reading, development of research question and hypothesis, lab work,
supervisor meeting/discussion and personal reflection on your progress.
1. 6 What skills will you gain or develop in this project? You will gain the
following laboratory skills in bacterial culture and identification techniques.
• Bacterial growth and enumeration
• Purifying bacterial culture
• Identification of bacteria (Gram-staining, motility, catalase, oxidase)
• Bacterial growth curve assays (synergistic and antagonistic characteristics)
• DNA extraction, PCR/16S rRNA gene amplification and sequencing
• Bioinformatic analysis of 16S rRNA sequences
You will gain general skills in data analysis and interpretation, independent critical
thought and decision making, time management and planning and professional report
writing.
5
1.7 Relation to current Food Microbiologist or analysists
-Replicates foodborne pathogen identification in food industries
-Formulation of new food products in food industries
1.8 Health and safety considerations
You will be working with unknown bacterial isolates; organisms are less likely to
cause disease in humans. However, good laboratory and microbiology practice is
paramount. Health and safety is the responsibility of all staff and students involved in
this practical.
Students are thus reminded of general lab safety basics:
• Materials must not be placed in your mouth.
• Protective laboratory clothing must be worn, including lab coats and nitrile
gloves when handling microorganisms (or equivalent).
• Cover open wounds.
• Working space should be kept clean and cleared up.
• Wash your hands with anti-bacterial hand wash after handling microorganisms
and before leaving the laboratory.
Consider others:
• Fellow students
• Teaching & research staff
• Cleaning staff
• External contractors
• If you have any concerns or doubts about what you are being asked to do you
must communicate with an appropriate academic.
Students are also reminded of good microbiological practice:
• All cultures must be labelled with name of user, date and organism (not just
strain number).
• Contaminated material must be disposed of as soon as reasonably possible.
• No sharps (including slides & tips) should be left lying around after use.
• Remove gloves prior to writing or handling computers, phones or door
handles.
Students must keep items brought into the laboratory to a minimum.
6
Under no circumstances can any mobile phones, tablets or laptop computers be
brought into the laboratory.
Such items must be locked securely in the lockers provided (speak to the technical
staff if you have forgotten the required £1 coin).
Students are encouraged to read the following guidelines: Guidelines for Biosafety in
Teaching Laboratories http://www.asm.org/images/asm_biosafety_guidelinesFINAL.pdf
1.9 Summary of expected lab work
Students are required to follow the work schedule below and protocols in the
following sections, but you decide which probiotics drink, or probiotic organisms to
investigate and what control strains should be considered. Students are encouraged
to discuss with the academic team and record this in their lab books. All laboratory
sessions are mandatory for all students.
| Day | Date | Activity | page |
| 1 | 24th Jan Monday 10-1 |
• Media preparation, pouring plates, streaking, serial dilution, spread and drop plate practice; pipetting practice |
26,11- 14 |
AssignmentTutorOnline
7
| 2-5 | • Probiotics drink serial dilution and plating for enumeration of bacteria • Inoculate of probiotics in a MRS broth and incubate for O/N |
11-13 | |
| 2 | 26th Jan Wednesday |
• Observe colony morphology • Count colonies and determine CFU/ml for serial dilution samples • Streak O/N grown probiotics on MRS agar |
12-15 |
| 3 | 28th Jan Friday |
• Observe colony morphology • Isolate and streak further on MRS agars and check for purity • If you have a pure culture, perform Gramstaining |
12-15 |
| 4 | 31st Jan Monday |
• Isolate and streak further on MRS agars and check for purity • Gram-staining • If you have pure culture, perform biochemical tests (catalase and oxidase) and API 50 CH • Extract DNA and 16S rRNA gene amplification PCR- O/N |
1-18 |
| 5 | 02nd Feb Wednesday |
• DNA gel electrophoresis • PCR product clean up and prepare the sample for sequencing • Set O/N culture for growth curve |
18-22 |
| 6 | 04th Feb Friday |
• Growth assays- monoculture and co-culture (triplicates) • Set O/N culture for Antagonistic plate assay |
23 |
| 7 | 07th Feb Monday |
• Bioinformatics; 16S rRNA gene sequence analysis |
|
| • Synergistic and antagonistic plate assay | 24 |
8
| 8 | 09th Feb Wednesday |
• Prepare prebiotics and own food of choice (filter milk, soy milk, cocoa extract, mushroom extract, inulin or autoclave) • Design formulation (e.g., calculate volume and percentage of each prebiotics, etc.) • Set O/N culture for formulation |
|
| 9 | 11th Feb Friday |
• Growth Assay with new probiotics monoculture and co-culture • Timepoint assay (drop plate) |
23 |
| 10 | 14th Feb Monday |
• Own or specific activities • Survival assay |
|
| 11 | 16th Feb Wednesday |
• Own or specific activities • Survival assay |
|
| 12 | 18th Feb Friday |
• Own or specific activities • Survival assay |
1.10 Materials required
• MRS agar and broth (in excess; use regularly)
• Sterile 96 well microtitre plates
• Syringe filters
• Sterile 6 mm disks
• Membrane filters
• Anaerobic jars with packs
• Colony counter
• Multiskan Go (plate reader)
9
• API 50 CH • 16S rRNA primers
Strains:
• Lactobacillus casei Shirota (UoB strain) Positive control
• Lactobacillus acidophilus ATCC 4356
• E. coli -Negative control
2. FLOW DIAGRAM OF WORKFLOW
10
| Enumeration | CFU- serial dilution, Miles and Misra |
| Isolation |
| Broth inoculum O/N and streaking & Membrane filtration plating |
Purification of culture
| Phenotypic identification |
| Colony morphology, Gram staining |
| Biochemical identification |
| Catalase, oxidase and API50 CH |
Identification
| Molecular ID 16S rRNA |
| Bioinformatics analysis of 16S RNA gene |
| Growth assays |
| Monoculture and co-culture |
Antagonistic plate assay
Formulation
Survival assay
Commercial Probiotics
11
3. PROTOCOLS
3.1 Enumeration
Spread plate: Carry out dilutions of each commercial probiotics sample and
spread on MRS plates
To determine the number of viable cells in each sample, students should prepare a
series of dilutions and spread a sample of appropriate dilutions onto MRS plates.
Figure 1. Serial dilution technique
1. In preparation for the serial dilutions, 900 μl of diluent (PBS saline) should be
added to each sterile bottle.
2. Starting the Sample 1, serial dilutions should be carried out by adding 100 μl
commercial probiotics sample added to 900 μl diluent to form 10-1 dilution;
100 μl from 10-1 added to 900 μl diluent to form 10-2; 100 μl from 10-2 added to
900 μl diluent to form 10-3; and so on.
3. Plate out 100 μl of 10-5, 10-6, 10-7 & 10-8 dilutions onto separate MRS plates.
To do this, 100 μl of the dilution is added to the centre of the plate and spread
over the total surface with a sterile spreader.
4. Perform triplicates (3 plates per dilution) for each dilution and take average
CFUs/ml (12 plates per sample)- you are doing two samples)
5. Incubate the plates at 37°C for 24 hours, count the colonies in each plate, and
calculate the Colony Forming Units (CFU) per millilitre. Note: Only 0.1 ml (100
μl) should be plated from each dilution on one plate.
12
6. Note: some of the plates may have too many colonies to count – this should
be recorded as TMTC or TNTC (Too many to count or too numerous to
count). It may be that the plate contains confluent growth – if so this should be
noted.
7. Taking into consideration the dilution of the sample in the serial dilution
procedure and the volume spread onto each plate, each student should
determine the number of bacteria in 1 ml of the original sample (expressed as
CFU ml-1, where CFU = colony-forming unit).
8. Calculate the number of bacteria in 1ml of each sample.
Calculation: CFU/ml=
3.2 Enumeration: Drop plate method/ Miles
| and Mishra plating (Herigstad et al., 2001 and Montana state Uni protocol) |
method: |
• 10 µl from the above serial diluted samples should be dropped on MRS agar.
• A MRS plate is divided into 4 quarters and 5X10 µl of 10-1 dilution is placed in
one quarter of the plate (5 replicates of each sample in each quarter) as
shown in figure below (Fig-2).
• Incubate the plates at 37°C for 24 hours and count the colonies in each plate
and calculate the Colony Forming Units
(CFU) per millilitre. Note: Only
0.01 ml (10 μl) plated from each
dilution on one plate.
• The same is repeated for 10-2 and so
on until 10-8
| CFU=No. of colonies X Dilution Factor (DF) If you get 311 colonies from 103 dilution, for 0.1 ml cells plating CFU/0.1ml = 311 X 10 3 =3.11 X 10 5 CFU/ml= 3.11 X 10 5 X 10 = 3.11 X106 |
| CFU=No. of colonies X Dilution Factor (DF) If you get 3 colonies from 103 dilution, for 0.01 ml (10 µl) cells in drop plate method CFU/0.01ml =3 X 10 3 CFU/ml= 3 X 10 3 X 100 = 3 X105 |
13
Fig-2- schematic representation of Drop plate
3.3 Isolation of Probiotic organisms
Broth inoculation:
• 2 grams (approx) of the commercial Probiotics food should be inoculated into
100 ml MRS broth and incubated at 37°C for overnight or 24 hours
• Spread 10, 25, 50 and 100 ul of the overnight culture on MRS agar and
incubated for 24-48 hours (in most of cases you will see colonies in 24 hours,
if not extend the incubation)
• If the colonies are not pure culture (mixed culture), pick up a single colony
and restreaked on MRS agar and purify them. This should be repeated until
you see pure culture (check with an academic staff).
• Finally, the pure culture is your strain for the project.
3.4 Streak plate method: Streak bacteria with sterile loops using the conventional
streak plate procedure
• Using a sterile loop, streak an inoculum of samples from the prepared cultures
or broth onto separate MRS plates/nutrient agar plates to determine the
phenotype of the bacterium. Each student should also streak control strains
provided on MRS agar.
14
Figure-3: Standard streaking pattern
• The initial inoculation will be done with a first sterile loop (L1) across one side of
the plate. Taking a fresh loop, a series of parallel lines are dragged from the
inoculum pool (L2a). Flipping the loop, the other edge can be used to draw
further parallel lines (L2b). A fresh loop can be used to complete the streaks
(L3a & L3b) ending with a squiggle into the centre of the plate.
3.5 Phenotypic Identification: Colony morphology
L2
L2
L3
L3
L
15
Figure 4. The various forms, elevations, and margins of bacterial colonies
3.6 Gram staining and Microscopic observation
1) Students should select representative colonies from their TSA plates and
carry out the Gram stain procedure as follows. Students should also prepare
slides with the positive control (Lactobacillus casei) and negative control (E.
coli) strains provided
2) Place a small drop or loop-full of sterile saline (PBS) on a clean glass slide.
3) With a sterile loop, touch a single bacterial colony. Emulsify the bacteria in the
saline drop and spread out to make a thin film on the microscope slide.
4) Allow to dry in air.
5) Heat-fix the bacteria on the slide by passing through a Bunsen flame two or
three times & allow to cool.
6) Stain the film by following this Gram’s stain method (gloves HIGHLY
recommended!):
| Reagent | Time on slide | Action following incubation |
| Crystal violet (primary stain) | 30 seconds | rinse with water & drain |
| Gram’s Iodine (Mordant) |
30 seconds | rinse with water & drain |
| Acetone/Alcohol (Decolouriser) |
5-10 seconds | rinse with water & drain |
| Safranin (Counter stain) | 30 seconds | rinse with water, drain, blot & air dry |
7) Examine the film under the oil immersion lens (X 100) of the microscope.
8) Note the colour, shape and arrangement of the organisms.
3.7 Simple Biochemical tests
Catalase test:
16
• Carry out the catalase test on any Gram-positive isolates as follows: (Modified
from the following SMI https://www.gov.uk/government/publications/smi-tp-
8catalase-test)
1. Place 4 to 5 drops of 3% hydrogen peroxide solution in a bijoux bottle for each
test strain, ensuring that you label all tubes
2. Carefully pick a colony to be tested with a
disposable loop
3. Rub the colony on the inside wall of the bottle
just above the surface of the hydrogen peroxide
solution
4. Cap the bottle and tilt it to allow the hydrogen
peroxide solution to cover the colony
5. Observe for immediate bubble formation, which
indicates a positive reaction
6. Pause here and discuss with the teaching team
Oxidase test:
The Enterobacteriaceae are an important group of Gram-negative bacteria. If the
unknown isolates have characteristics of Gram-negative bacteria and are straightsided rods, you should carry out an oxidase test as
follows:
NOTE: a positive control must be also tested to ensure
the assay is working
1. Cut a piece of filter paper in half, labelling each
piece. Place the pieces in a sterile petri dish and
apply the reagent solution (Remel™ BactiDrop™
Oxidase) https://www.youtube.com/watch?v=z1HsdAQO7m4&NR=1
2. Scrape some fresh growth from the relevant culture plate with a disposable
loop and smear onto the wet filter paper
| Fig 6- oxidase test |
Fig-5-Catalase test
https://mltgeeks.com/biochemicalcatalase-test-principle-and-interpretationsall-catalase-positive-organism/
17
3. Observe for colour change within 10 seconds. A purple colour within this time
indicates a positive reaction for the oxidase test
API CH (follow Manufacture’s instruction)
3.8 Genotypic Identificaiton: Genomic DNA extraction and 16S rRNA gene
amplification:
For rapid DNA extraction, the rapid lysis method described by Zhang et al. (2004)
will be used.
(Zhang et al. (2004) New Quadriplex PCR Assay for Detection of Methicillin and
Mupirocin Resistance and Simultaneous Discrimination of Staphylococcus aureus
from Coagulase- Negative Staphylococci. J. Clin Microbiol. 42 4947-4955.)
• Using a sterile pipette tip, a bacterial colony should be suspended in 50 μl of
sterile distilled water and heated at 99°C for 10 min.
• This suspension of lysed bacteria should then be centrifuged at 20,000 X g for
1 min 3 μl of the supernatant will be used as a template in each of the PCR
reactions.
• This suspension of lysed bacteria should then be centrifuged at 20,000 X g for
1 min 3 μl of the supernatant will be used as a template in each of the PCR
reactions.
16S Bacterial primers
3.9 Setting up 16S rRNA PCR reaction
Set one reaction per isolate and include one negative control with no template
5x master mix -25 l
Water -20 l
16s Forward primer 10 M -1 l
| 16s Rev primer 10 M | -1 l |
| gDNA template | -3ul |
| Total | -50 l |
18
PCR cycle parameters
Initial denaturation: 95 degrees- 1min
1. Denaturation: 94 C – 15s
2. Annealing: 50 C – 15s
3. Extension: 72 C – 10s
Repeat step 1-3 for 30 cycles Final
extension 72 C for 4 mins and
hold at 4 C
3.10 Agarose gel electrophoresis of DNA fragments
Important safety issues
• Ethidium bromide (within the agarose solution) is extremely TOXIC. It is a
MUTAGEN! Gloves must be worn when pouring agarose, applying samples
and during viewing and
• changed/discarded as soon as possible afterwards. Risk of Electric shock
from Powerpacks!
• Cover the electrophoresis tank with the lid before switching on.
• Switch off the power before removing the lid from the electrophoresis tank. UV
light can damage skin & eyes!
• Use of the gel documentation system should be supervised and students must
never look directly at UV light without full face protection.
• Prior to the class, a 2 % solution of agarose has been prepared in 1X TAE
electrophoresis buffer. The solution was boiled to dissolve the agarose and
allowed to cool to approximately 55°C. When cool, ethidium bromide was
added to a final concentration of 1 μg/ml.
• Gel loading buffer needs to be added to all samples to a final concentration of
1X. Such gel loading buffers typically contain a viscous component to allow
samples to sink into the wells and a dye, such as bromphenol blue, which
migrates in agarose gels to show progress of the procedure. However, the
mytaq red mix already contains these so students need not add this when
running PCR products prepared using this reagent.
• 5 μl of each of PCR product are typically loaded on an agarose gel although
more may be required if the product is to be purified for sequencing.
• In addition to their samples, students should also have a molecular weight
marker, which will be added to the gel as a reference to determine the
approximate size of DNA fragments. This marker already has gel loading
buffer added and 5 μl should be loaded on the gel.
• The comb should be removed from the agarose gel and 1X TAE added to the
electrophoresis tank, so that the gel is just submerged.
• Each sample can then be loaded into the wells by pipetting the sample into
the opening of the appropriate well – taking care not to stab into the well,
which may result in puncturing through the bottom of the gel.
In order to interpret the results, it is necessary to note the order in which the samples are
loaded to the gel!
19
Before switching on the electricity, students should ensure that the power cables are
attached to the power pack so that DNA will migrate towards the positive electrode.
If in doubt check with the teaching team.
The gel is “run” by applying an electrical current, between 80 & 100 V, for
approximately 40 minutes or until the dye runs halfway down the gel.
When complete, students should ensure that the power is turned off before removing
the lid of the electrophoresis equipment. The gel can then be viewed with an UV
transilluminator and gel documentation system. The teaching team will help students
receive a picture of their gel image.
3.11 PCR clean-up /gel extraction of PCR products (Manufacturer’s protocol is
shown below)
20
Bioline Isolate II PCR clean up protocol
Note: Step 5, elute DNA with 20 µl of sterile water.
21
Note: Step 5, elute DNA with 20 µl of sterile water.
3.12 Sample preparation for sequencing 16S rDNA gene
Students will need to first know the size of their PCR product as determined by
agarose gel with comparison to the DNA size marker (commonly referred to as the
kb ladder).
The concentration of the PCR product should be determined using the nanodrop.
1. A single microliter should be placed onto the measurement pedestal
22
2. Gently close the sampling arm.
3. Initiate the spectral measurement using the operating software as directed by
the teaching staff.
4. Students should note the concentration of their sample (ng/μl) & the 260/280
ratio (which gives an indication of the quality of the sample).
5. When the measurement is complete, open the sampling arm and wipe the
sample from both the upper and lower pedestals using a soft laboratory wipe.
Calculation:
Note: The Cambridge DNA sequencing facility want 10 μl of PCR product with a total
amount of 20 ng per 100 bp (0.1 kb) of product.
Amount of PCR product to add (ng) = Approx size of PCR product (bp) X 20ng
100 bp
The volume of PCR product to add (µl) = Amount of PCR product to add (ng)
The concentration of PCR product (ng/µl)
3.13 Bacterial growth assay: Bacterial growth mono and co-culture assay
1. 2 to 3 colonies of each organism (Organism A and B seperately) should be
inoculated into 10 ml MRS broth and incubated at 37°C for overnight at
shaking incubator.
2. Next day-check the optical density (OD) of the overnight cultures (A & B) at
23
600nm using spectrophotometer. Use fresh sterile MRS broth as a blank for
600nm calibration. 900 ul of fresh MRS broth should be taken in a cuvette and 100
µl of overnight culture A should be mixed and checked the optical density.
Note: do not put the culture from the cuvette back to your culture tube.
Discard the cuvette with the culture appropriately. Do the same for B.
3. For example, if you get 0.0512 OD for 900 µl MRS broth +100 µl O/N culture;
then multiple by 10 for OD for 1 ml;
0.0512 X10= gives the OD for 1 ml,
which is 0.512 (O/N cells OD)
4. Now you prepare 3 ml of MRS broth with
0.1 OD cells for each culture growth
curve; calculate how much cells
required for 3 ml
5. So, according to the above calculation, take 0.58 ml of O/N culture + 2.42 ml
of MRS broth in a 25 ml universal tube. That should give you 3 ml of 0.1 OD
cells.
6. Prepare your 96 well plate for growth curve assay, 3 wells for each sample.
Add 200 µl 0.1 OD culture into the respective wells.
For example wells A1, A2 and A3 for your organism A (triplicates), well B for
organism B and wells C for the organism (A+B co-culture).
For A+B well add 100µl of organism A and 100 µl of organism B.
Each student requires 9 wells. 4 students share one 96 well plate (see below).
S1- Student 1, S2-students 2, S3- students 3 and S4- students 4
Fig-7- Schematic representation of Microtitre plate
| Required cell density X required volume Stock/ or O/N cell density 0.1 OD X 3 ml =0.58ml 0.512 |
| A1 A2 A3 |
| B1 B2 B3 |
| A +B A+B A+B |
| A1 A2 A3 |
| A +B A+B A+B |
| B1 B2 B3 |
| A1 A2 A3 |
| B1 B2 B3 |
| A +B A+B A+B |
| A +B A+B A+B |
| B1 B2 B3 |
| A1 A2 A3 |
S1
S2
S3
S4
24
7. The plates should be incubated at 37°C for 48 hours and read the optical
density at every 30-minute interval for 48 hours at 600nm.
3.14 Bacterial growth Synergistic and Antagonistic plate assay
1. A single colony of probiotic organisms 1 and 2 are separately inoculated into 5
ml MRS broth and incubated at 37°C for overnight at shaking incubator
2. The optical density of overnight culture should be checked at 600nm using a
spectrophotometer, use MRS broth for calibration.
3. Prepare 0.1OD cells for your test organisms
4. Spread 0.1 OD600 cells (organism A) onto MRS/MHA plate with a sterile
cotton swab. The culture should be spread in 3 directions.
http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test
_documents/Slide_show_v_5.0_EUCAST_Disk_Test.pdf
Fig 8- 3 ways streak
5. Allow the plates to dry for 5 minutes at room temperature.
6. Place 3 sterile filter paper disks on top of
the agar (as shown in the picture using
sterile forceps and inoculate 100 µl of
(organism B) 0.1 cells)
7. Leave the plate with the disks for 5
minutes to adsorb at room temperature (do
not invert the plates).
8. The plates should be inverted and
incubated at 37°C for 24 hours
9. Repeat the same procedure for organism B on the plate and organism A on
the disk.
10.Using a ruler, record the diameter of the zone of inhibition in millimetres.
| Fig 9- Antagonistic plate assay |
| Organism A lawn Organism B on disk Organism B lawn Organism A on disk |
25
3.15 Formulation and Survival test
Enumeration after 1,2,3,4 day of formulation
4. KEY RESOURCES:
• Zotta et al., (2013) Temperature and respiration affect the growth and stress
resistance of Lactobacillus plantarum C17,.Applied Microbiology, 115, 848858
• Schifano et al (2021), Leuconostoc mesenteroides Strains Isolated from
Carrots Show Probiotic Features.Microorganisms,9, 2290
• Davis, Catherine. “Enumeration of probiotic strains: Review of
culturedependent and alternative techniques to quantify viable bacteria.”
Journal of microbiological methods vol. 103 (2014): 9-17.
• Karami, Sahar et al. “Isolation and identification of probiotic Lactobacillus from
local dairy and evaluating their antagonistic effect on pathogens.” International
journal of pharmaceutical investigation vol. 7,3 (2017): 137-141.
• Janda, J Michael, and Sharon L Abbott. “16S rRNA gene sequencing for
bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls.”
Journal of clinical microbiology vol. 45,9 (2007): 2761-4.
• Song, A.AL., In, L.L.A., Lim, S.H.E. et al (2017). A review on Lactococcus
lactis: from food to factory. Microb Cell Fact 16, 55 (2017).
• Kerry et al., (2018). Benefaction of probiotics for human health: A review,
Journal of Food and Drug Analysis, Volume 26, Issue 3, Pages 927-939.
• Kechagia, Maria et al. (2013). “Health benefits of probiotics: a review.” ISRN
nutrition vol. 2013, 481651.
• Gueimonde and Salminen (2006). “New methods for selecting and evaluating
probiotics.” Digestive and liver disease : official journal of the Italian Society of
Gastroenterology and the Italian Association for the Study of the Liver vol. 38
Suppl 2: S242-7.
• Bermudez-Brito et al., 2012, Probiotics mechanisms of action, systematic
review, Annals of Nutrition and Metabolism, Vol.61, No. 2
• Rastelli, Marialetizia et al (2018). “Gut Microbes and Health: A Focus on the
Mechanisms Linking Microbes, Obesity, and Related Disorders.” Obesity (Silver
Spring, Md.) vol. 26,5: 792-800.
• Herigstad, B et al. “How to optimize the drop plate method for enumerating
bacteria.” Journal of microbiological methods vol. 44,2 (2001): 121-9.
doi:10.1016/s0167-7012(00)00241-4
• Pelinescu et al., (2015) Isolation and identification of some Lactobacillus and
Enterococcus strains by a polyphasic taxonomical approach.Romanina
Biotechological letters.14 No2, pp 4225-4233
• Matejčeková etal., (2016), Characterization of the growth of Lactobacillus
plantarum in milk in dependence on temperature.Acta Chimica Slovaca, Vol 9,
No.2, pp 104-108
• Yang, et al., (2018) Infuence of culture media, pH and temperature on growth
and bacteriocin production of bacteriocinogenic lactic acid bacteria.AMB Expr
8:10
26
Appendix- 1
Preparation of agar plates
You are given a sterile molten agar
• Cool the agar to 45°C
• Using an aseptic technique, pour the agar into sterile Petri dishes
• Pour about 20 ml agar in each plate
• Plates should be allowed to air dry (with the lid at an angle as shown below) before the cover
is fully replaced. Make sure your plates are dried.
Fig 10 : pouring plates
- Assignment status: Already Solved By Our Experts
- (USA, AUS, UK & CA PhD. Writers)
- CLICK HERE TO GET A PROFESSIONAL WRITER TO WORK ON THIS PAPER AND OTHER SIMILAR PAPERS, GET A NON PLAGIARIZED PAPER FROM OUR EXPERTS
