Procedures for the workshop

1. Media supplements
Environmental bacteria and fungi:
Minerals (from autoclaved soil)
Starch and sugars (rice milk and potato flakes or starch)
Yeast extract as source of amino acids (Weißbier or backer’s yeast autoclaved)
Test to inhibit bacteria growth and allow fungi selection: germination water red beans (Sterile
filtrated)
Media recommended for fungi:
PDA: Potato Dextrose Agar.
From https://microbiologyinfo.com/potato-dextrose-agar-pda-principle-uses-composition-
procedure-and-colony-characteristics/
Potato infusion 200 g
Dextrose 20 g
Agar 20 g
Distilled water 1 liter
Note: 200 g of potato infusion is equivalent to 4.0 g of potato extract.
In percentages: PDA contains 2% agar, 3% Dextrose, 0.4% of potato starch (Kartoffelnstärke). To inhibit bacterial
growth it is recommended to lower the pH to 3.5. or add chloramphenicol.
Human bacteria:
Egg yolk (fatty acids and iron)
Sugars (sugar cane)
Amino acids (Yeast extract)
Salt (0,5% salt)
Agar → 1,5%
Food bacteria
Milk (For Lactobacillus spp)
Mannitol for Acetobacter spp (Source: Sugar-free sweeteners like Xylitol, Maltitol, Isomalt)
Project: From Delicacy to Poison: Misunderstood Microbes
Objectives:
• Try to make visible the toxins / secretions of fungi in the media.
• Try to make visible the hyphae INSIDE the media, to distinguish from Fruit body to hyphae
(Filaments of the micelium from the fungi)
• Show the wonders of a fit Immune system and microbiome in protection of host: Show the
pepper and peanuts growth.
• Determine the times and quantities we will need to show the effects of:
◦ High concentration of salt (Yeast in flour dough)

2. ◦ High acidity (yeast bubbling in vinegar concentrations maybe in apple juice?)
◦ Making Yogurt from full milk and lactose free milk (Show that lactic acid is made from
lactose and break the concept of lactose-free yogurt)
• Show the concept and importance of positive and negative controls.
• Prepare slides from Camembert or Brie cheese (For hyphae coloration by each of the
participants).
• Show dangers of blown cans and homemade canning (Botulinum toxin by Clostridium
botulinum).
• Show the dangers of bad prepared milk products in fridge and consequences.
• Show the growth of human bacteria in high concentration of salt (Staphylococcus aureus)
• Isolate a gram negative and a gram positive to show their membranes resistance using 3%
KOH solution with toothpicks and a glass slide.
Show the future of microbiology: Bioinformatics for analysis of Metagenomics, genomics,
transcriptomics, metabolomics and proteomics.

3. Practical Microbiology (Beginners to Intermediate level)
Title: Misunderstood Microbes TM
: From Delicacy to Poison.
Description: Transforming simple food to delicacy requires the work of “miraculous”
microorganisms. However they need to be the “chosen” ones. In this course, we will explore the
world of microbes through their roles in our food production, by highlighting the relevance of
microbiological concepts to prevent our delicacies turning into poison.
Presented by Dr. Luisa F. Jiménez-Soto and made possible by Bio.Kitchen.
Start time: 9:00 (sharp/on time).
Estimated end: 18:00
Knowledge required: Basic laboratory safety concepts.
Course Objectives
During the course we will explore basic microbiological concepts through the following:
1. Microbes in food and their metabolism producing alcoholic beverages,vinegar and milk-derived
products.
2. When microbes growth where they should not: Toxin production and impacts caused by modern
human technologies.
3. Relevance of microbes protecting our body: Making visible all what could attack us and does not
(usually).

4. Misunderstood Microbes: From delicacy to poison.
Objectives
1. Microbes in food and their metabolism producing alcoholic beverages,vinegar and milk-derived
products like Yogurt, Kefir, Cheese (old cheese).
2. When microbes growth where they should not: Toxin production (fungi growth vs toxin
distribution) and growth caused by modern human technologies(Listeria monocytogenes)
3. Relevance of microbes protecting our body: Making visible all what could attack us and does not.

5. Preparing Media and solutions
Potato-Dextrose-Media (For Fungi growth)
2 grams of Agar agar
3 grams of sugar
0,4 grams of potato starch
Fill to 100 ml volume with distilled water
Autoclave. Once the media reaches around 56°C, add the antibiotic and mix properly.
Pour approx. 20 ml of media per petri plate.
Let cool down and store at 4°C until use.
For other bacteria, use the standard Media LB, Endo and MRS broth / Agar as recommended.
Experiments
General Objectives
• Show that microorganisms are everywhere.
• Show the importance of controls in experimental set up.
• Show the relevance of metabolism knowledge for food safety.
• Document everything for use in further educational programs and Open Source projects.
Production of gas
Objective(s):
• Show the relevance of Safety Lids (Normal vs bulged)
• Show that certain bacteria can produce gas, while others cannot.
Cultivate Lactobacilli in MRS Broth and agar
Background
First, we will need to isolate Lactobacilli. For this, prepare MRS agar using MRS broth and 1.5%
agar agar. Lactobacilli tolerate better acidic conditions than Streptococci. Using a pH 5.5 to 6.5 will
allow to reduce the chances of isolating Streptococci from the yogurt sample. Other mechanism for
selection is salt. Streptococci are extremely sensitive to salt (NaCl) concentrations starting at 5%
and above.1
The resulting media will be MRS agar with 5% NaCl. Measure pH. Once single bacteria are
isolated, grow in MRS broth and perform the gas production test. If positive, it is not Lactobacillus
spp., but Leuconostoc spp. As sample use diluted natural yogurt in sterile distilled water.
1 https://jcm.asm.org/content/jcm/21/2/247.full.pdf

6. Protocol for Isolation of Lactobacilli from Natural Yogurt2
1. Take 100 µl of yogurt into 900 µl of destilled water (initial solution, or dilution 100
).
2. Mix by pipetting.
3. From the initial solution, take 100 µl into 900 µl of water. This is the dilution 10-1
.
4. Take 100 µl of dilution 10-1
and place the aliquote on the MRS agar plate.
5. With a sterile Drigalski cell spreader, spread the bacterial suspension on the agar. Take care
of working sterile the whole time.
6. Close lid of petri plate and turn it upside down in the incubator. Bacteria should grow at
mesophilic termperatures (37°C).
7. Incubate for 24-36 hours.
8. Select colonies that are white, slightly shiny, of about 1 to 2 mm diameter, concave
Identification of Lactobacillus spp.
• By PCR, using primers from https://www.ncbi.nlm.nih.gov/pubmed/15476976 or
https://academic.oup.com/femsle/article/214/2/271/611104
• Gram staining with microscopy analysis if possible.
Test for gas production by Lactobacillus spp.
1. Grow isolated Lactobacillus spp using the isolation method “streak plate” on agar.
2. Take one colony of Lactobacillus spp with a sterile tip and place it in 5 ml MRS broth.
3. Incubate if possible under shaking conditions during 24 hours at 37°C.
4. Verify growth by verifying the optical density at 600 nm.
5. Once the liquid culture reaches OD600 between 3 and 5, take 1 ml to inoculate a new MRS
broth tube.
6. Melt about 10 g of stearin and pour it onto the liquid media. Stearin is hydrophobic and
should be lighter than water, so it should remain on the surface of the liquid media. Pour
until the stearin created a 1 cm thick plug.
For the test, repeat same procedure with positive (E. coli in DIY broth) and negative (MRS broth
sterile) controls
DIY Broth
1. Suspend 2 grams of Meat broth (2%) (and 0.5 g yeast extract (0.5%) if it is not contained
already in the meat broth) in 100 ml distilled water.
2. Autoclave and pour 5 ml into 10 ml tubes.
3. Store at 4°C until use.
Cultivating fungi for its association food.
Background
Fungi are eukaryotes, meaning that they have isolated their DNA in a membrane limited organelle
called nucleus. Additionally, other cellular functions and reactions have been isolated in different
organelles. Animals and plants are as well eukaryotes. However, fungi distinguish from other
2 Today 11.9.2019 Making Yogurt: 600 ml milk, 60 g natural yogurt, in YogoBereiter for 6 hours. All materials were
left at least 2 h RT to increase bacterial activity and possible reduce time of incubation.

7. eukaryotes on their chitin-containing cell wall. Plants have as well a cell wall, but it is made of
cellulose (main component of paper). Chitin is only found outside of fungi in the exoskeleton of
arthropods (spiders, insects, crabs), and functions as a structural reinforcement of the “body” of the
cell or arthropod.
Fungi are able to grow in two (sometimes it has been classified as three) different forms: Yeast and
molds. Yeast are usually unicellular while molds are composed of many cells connected through a
septum. The multicellular organism shows signs of rudimentary specialization (asexual and sexual
cells).
One yeast species is one of the oldest microorganism with a pivotal role in human civilization and
culture: Saccharomyces cerevisiae . Known as well as baker’s yeast or brewer yeast, it is
responsible for the fluffiness of bread and the alcohol production in wine or beer. It is found
ubiquitous (meaning everywhere): environments, gut of animals, etc.
Mold type fungi have as well played a role in human civilization. However not always their
interaction with humans has been beneficial. Although mold species Penicillium spp have open the
door to the use of antibiotics, others have played a role in magic, health and myths as hallucinogens.
Here we will explore fungi in food, their role in creating alcoholic beverages by creating alcohol,
their use in bread with their capacity of producing gas, the creation of some of the most delicious
cheeses and their contrast with their ability of producing one of the most lethal toxins: flavotoxins.
Yeast experiment I: Yeast burping conditions.
Objective
• Explore and determine the conditions in which the baker’s yeast produces gas from their
metabolism of wheat flour.
• Learn when sterile work is really necessary and not.
Procedure
1. . Activate the dry yeast in a 10 ml of a mixture of warm milk and sugar (0.5% sugar in milk)
for 15 min.
2. Prepare the test tubes with 2X concentrated 3
test conditions in milk and wheat.
1. 1, 2, 3 4 and 5% salt.
2. 4°C, 25°C, 37°C and over 70°C.
3. Aliquote in each tube 500 µl of the activated yeast and mixed with pipettes.
4. Incubate at room temperature for 30 min.
5. Note the effects of treatments compared with positive and negative controls.
3 These concentrations need to be tested. We need to be able to pipette the suspension.

8. To make visible the fungi micelia
Background
After searching sources for the best way to see fungi in wet preparations, I found the following
information:
“Prepare a solution of lactophenol cotton blue. Weigh out 0.05 grams of cotton blue dye using a
sensitive weighing scale. Mix the dye with 20 milliliters of distilled water in a test tube and leave it
overnight. Put on gloves before finishing the stain preparation the next day. Place 20 ml of
lactic acid into a beaker and mix it with 20 g of phenol crystals. Stir until the mixture is dissolved.
Add in 40 ml of glycerol and mix. Then strain the cotton blue solution through a piece of filter
paper into the beaker and mix thoroughly.”
(Source: https://sciencing.com/stain-chitin-8123067.html, September 11, 2019)
Optional stains to Lactophenol Cotton Blue staining:
Glycerol (0.25%) in iodine solution. The iodine-glycerol as an alternative to LPCB can also be
used. (Source: ResearchGate Discussion
https://www.researchgate.net/post/What_safe_alternatives_are_there_to_lactophenol_cotton_blue_f
or_fungal_staining )
As a result, I decided to order a normal dye for fabric.
Fabric dye for fungi observation
Weight 0.05 g of dye, disolve in 20 ml distilled water. Leave overnight. Next day and solve in
distilled water containing 40% glycerol. Test another glycerol and dye concentrations for the
observation of fungi. Similarly, test different concentrations of acetic acid .4
.
Note: It is possible that the aromatic rings of the phenol have a stronger effect on the visibility of
the contrast of the hyphae. It needs to be tested if we find a good replacement for phenol.
4 Cotton blue is an aniline dye. I could not find a natural source, so I ordered synthetic aniline blue dye for clothes in
powder and we will use it to make the staining solution. All sites with LPCB information claim that phenol gives a
better contrast. I am almost sure that phenol is not available in the lab. However, I saw a lot of glycerol, which shares
some of the optical qualities of phenol. I just need to figure out what concentration of glycerol will work better. We will
start at 0.25%. Since we probably will not have filters for sterilization, we need to make very small amounts of the
solution. Glycerol is an excellent source of food for “fluffies”. About the lactic acid, I am not really convinced on the
“reasons” the websites offers for its use in the solution. I will keep searching. If I do not find something, I will try to
replace it with vinegar.