Food Dye, Watercolors, testing on Bacterial celullose

 

Started with a thick wet sheet of bacterial cellulose, I washed it with lukewarm water and added some watercolors.

Then added some food dye to next to the watercolor, I purchased this food dyes from Amazon, there were very inexpensive were absorbed by the bacterial cellulose very fast.

I added some glow in the dark pigment, I do not recommend the brand as I did not see any results. I have purchased glow in the dark pigment that would result in very vibrant colors. Unfortunately this one was not visible at all. Also, the box states that is highly toxic.

Lastly  I added some gold flakes just to see if they would stick to the cellulose, they did!

Wet on the left , Dry on the right.

***The only issue I'm encountering is the that the sheets are drying and becoming very stiff.

Basics. YPS media, recipe, yeast info

 YPS

In order to grow a healthy SinScoby YPS media is needed. YPS is a type of Yeast media, the acronym stands for Yeast Peptone Sucrose.

• 5g yeast extract

• 10g peptone

• 10g sucrose or sugar

• 500ml distilled water

• First step measure everything, then put all the ingredients in a glass bottle, shake it until no lumps of dry ingredients are noticeable.  Remove the bottle cap wrap it in foil, top the bottle with a piece of foil, add the autoclave tape as it is a process indicator for steam sterilizers. Put the bottle and bottle cap in the autoclave (machine that applies high-temperature steam to sterilize objects).

Distilled water

Peptone, this one is animal origin

Since there was no sucrose available in the lab we used regular sugar.

Though autoclave tape may have changed color during the sterilization process, this does not necessarily mean that an item is fully sterile. The tape will change upon exposure to the desired temperature it is indicated for. However, for proper steam sterilization to occur, the entire item needs to reach and maintain a temperature of 121 degrees Celsius for 15 to 20 minutes to ensure the items' sterility.

Note that autoclave tape and the indicators do not prove that the sterilization process has killed organisms. Instead, they demonstrate that the autoclave has reached a temperature of 121 degrees Celsius. Environmental, Health and Safety recommends that you do not use autoclave tape as the only indicator of decontamination or sterilization.

When using autoclave tape, attach it to the exterior of the item you want to sterilize. Depending on the kind of autoclave tape you use, the tape will change to a specific color.

Yeasts are eukaryotic microorganisms whose genomes have been comprehensively studied and some have been sequenced. They are relatively easy to grow under laboratory conditions. Moreover, despite their small genome size, they display cellular features and processes that are highly conserved amongst most eukaryotes. For instance, they have membrane-bound organelles, cytoskeleton, nuclear DNA, and transcription mechanisms that are similar to those found in higher eukaryotes. Furthermore, yeasts have many well-characterized secretory proteins and pheromones. Several yeast genes involved in protease processing and secretion have also been identified. Thus, yeasts can be used for several eukaryotic gene and protein studies with the aid of suitable molecular biology tools. Some applications for yeast cultures include synthesis of protein expression systems, the study of specific gene or protein functions, and the analyses of novel protein interactions.

YEAST MEDIA OPTIONS

1. YPD medium
A nutritious medium available in liquid (broth) or solid (agar) forms for the growth and propagation of yeast cultures. It primarily contains of bacteriological peptone, yeast extract, and glucose. This medium is non-selective for Candida, Pichia, Saccharomyces, and Zygosaccharomyces.

2. Yeast Synthetic Drop-out Medium Supplements
The selection of plasmids in yeast is usually based on the use of auxotrophic mutant strains, which cannot grow without a specific medium component (like an amino acid, purine, or pyrimidine). Transformation with a plasmid containing the mutated gene enables the transformant to grow on a medium lacking the required component. Thus, drop-out medium supplements can be used to select transformed yeast cultures.
Please refer to the selection guide below for further details.

3. Yeast Nitrogen Base (YNB with or without amino acids)
Yeast Nitrogen Base is a highly-referenced growth medium used for the cultivation of yeast. This nutrient-rich microbial medium contains nitrogen, vitamins, trace elements, and salts. It is suitable for use in classifying yeasts based on amino acid and carbon requirements. With an added carbon source, YNB with amino acids can be used for susceptibility testing of fungi.

Source

Yeasty boys

 Added some dry yeast. on top of the a growing scoby. I was inspired by the traditional painting with yeast. I wanted to use the powder as a way to observed how the yeast would react to the scoby environment. I added dry instant yeast,

Results, nice vibrant orange

Final result

Re-integrating a dry scoby into a new culture

 

November 10

November 15

Venus

 

Synscoby Co Culture

Day 0 (Wednesday PM) November 30th

1. Inoculate K. rhaeticus from glycerol stock in 5mL YPD + 1% cellulase
2. Place in shaking incubator at 30˚, 250rpm for 3 days
3. Streak S. cerevisae on YPD plates so fresh when inoculating on day 3.

To avoid contamination the YPD + Cellulase 1% were added into a centrifuge tube in the hood, then added to smaller to tubes with snap off caps.

Cellulase                                                    YPD

Outside of the hood we added the K.Rhaeticus with tooth picks into 30 ml tubes.

There were a total of 6 tubes so we can start co coulture of (3x BY4741p + 3 x Venus Ura)

Streaking new fresh yeast from the October batch into Agar plates 

Isolating yeast at this point is as simple as taking a very small amount of your culture and rubbing (streaking) it on to an agar plate. Because of the stable, non-liquid agar medium, once streaked, single colonies of microbes are essentially stranded by themselves.

After a few days or weeks (depending on incubation temperature and microbe population), they’ll multiply and grow large enough to be seen with the naked eye. At that point, it’s a matter of selecting the colonies you like and growing them up to larger amounts.

It’s impossible to stress enough how important cleanliness and sanitation are at this point in the process. If you truly want a pure strain, you need to ensure you’re not contaminating what you’re trying to isolate with other cultures.

Day 2 (Friday PM) December 2nd

1. Inoculate S. cerevisiae from plate in 5mL YPD
2. Place in shaking incubator at 30˚, 250rpm for 1 day

Day 3 (Saturday) December 3rd

1. Centrifuge K. rhaeticus at 3,220 g  (5119 rpm) for 10 minutes. 
2. Resuspend K. rhaeticus cells in YPS to OD600=2.5. This helps to remove cellulase.
3. Dilute S. cerevisiae in YPS to OD600 = 0.01
4. Combine K. rhaeticus and S. cerevisiae in 50mm petri dish, inoculating K. rhaeticus 1/50 and S. cerevisiae 1/100 in fresh YPS-optiprep. Final volume should be 15mL.
5. Incubate at 30˚ for 3 days under static conditions. Do not move co-cultures while growing or BC layers will come apart.

*****

YPS

• 5g yeast extract

• 10g peptone

• 10g sucrose

HS Recipe x 500 ml

• 2.5g yeast extract

• 2.5g peptone

• 1.35g sodium phosphate

• 0.75g citric acid

• 10g sucrose

• 500ml distilled water

**Snap off cap works best for K. Rhaeticus growth 

K.Rhaeticus

Growing critters ins Hestrin Schramm HS {brown media)  (look recipe)

K. Rhaeticus (bacteria) :  HS (hestrin schramm)  30c

HS Recipe x 500 ml

HS 

• 2.5g yeast extract
• 2.5g peptone
• 1.35g sodium phosphate
• 0.75g citric acid
• 10g sucrose
• 500ml distilled water

---------------

Pour from the stock to a separate tube is done in the sterile hood 

Streaking from Plates. with the microbes from (Part1) : 

Taking a loopful of microbe culture (a colony) from the plates and putting them in Media

 

October 25 LEFT                                       November 2nd RIGHT pellicle is visible - grew in 30C shaker

   

November 2nd

We run a test to see which tube top worked better a snap off cap and a screw top

The Snap off seems to be better as it lets more air come in

November 4th - moving forward we will only use Snap off tops

Nov7 - Testing to see if the K. Rhaeticus we took from the frozen stock will grow on HS media

We just took a bit from the frozen stock that we put in the -80C

Growing the pellicle in motion or still. Both will be growing at 30C, One in the shaker and one in the incubator

Nov 14 , The one on the shaker grew, the one in the incubator did not grew.