Lab 8 Production of beer

To learn:

Glycolysis is the metabolic pathway that converts glucose into pyruvate.

Fermentation in food processing typically is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions. Fermentation in simple terms is the chemical conversion of sugars into ethanol.

Material: germinated barley seeds, stainless stockpot, malt extract, yeast.
Method:

• Clean and sterilize everything before starting to make beer. 
• Heat up 2 gallon of water in the large pot. 
• Pour a bag of crushed grains into a grain bag. 
• Soak the grain bag in the hot water, boil for 25 min to activate enzyme.
• Life the bag, drain the water out without squeezing, discard grain. 
• Add three kind of sugar: malt extract, munich extract and pale extract.
• Stir constantly till all sugar dissolved. 
• Hops are added to the mixture to balance the sweetness. 
• Keep boiling and stirring, to kill the enzyme because we don't want to fermentated all malt. 

What's learned: there are 4 gredients are needed for making beer
Yeast
Hops
Malt
Water

The longer the mixture is cooked, the darker the solution is, however, the less achohol contained. The reason for this is sugar also get cooked, it cannot be fermentated. 

The purpose for boiling is: 
activate enzyme
deactivate enzyme
sterilization
precipitate protein, so the protein from grains can be removed. 

The next day, no fermentation happened. 
Possible reason:
1. temperature too low
2. yeast culture is not good
3. choloride in water inhibit yeast growth
4. sterilization solution left in the 6 gallon container may also inhibit yeast growth. 
We will inoculate another tube of yeast and try to make it grow.

Lab 6 Continuation of Neurospora genetics

Objective:
1. Examine perithecia, asci and ascospores of Neurospora.
2. Try to pick random perithecia and ascospores under dissecting microscope.
3. Try to observe the protein localization under fluorescence microscope.
4. Get a rough image for different Basidiomycetes morphology.
The purpose for this exercise is to get a basic idea of Neurospora sexual life cycle and structure for different sexual reproduction stage.

Method:

• 2 week old Neurospora mating plate, find shot perithecium or perithecium on plate (black beads)
• Clush perithecium on slide, observe the ascospores released from perithecium.
• Transfer some free ascospores on water agar plate.
• Incubate for one week.
• Pick ascospores from the water agar plate to tubes.
• Incubate at 4 degree to get hydrated
• Heat shock at 65 degree for 45 min to kill contaminated conidia.
• Transfer the ascospores to medium with nutrition, ascospores should germinate.

Result:
1.Asci and ascospores are observed with clush perithecium.
Neurospora is heterothallic, it requires different mating type for sexual reproduction. Opposite mating type hyphae mate to produce perithecia. At maturity, many asci are developed within perithecia. Each ascus contains 8 ascospores. Under scope, there are intact ascus and ascospores released from broken asus. However,Pro-perithecium has no content.

2. Premature ascus with 8 ascospores.
Usually, after ascospores matured,ascospores are pigmentated. Four of them are dark color, the other four are light green, which perfectly reflect genetic linkage.

3. Observe SMRP10 mutant nuclei under fluorescence microspores.

One of the feature for Neurospora is it has multinuclei. In this mutant, histone protein which is the most abundant protein in nuclei is tagged with GFP. Histone protein can help to localize nuclei. 

4. Observe different Basidiomycete morphology.
The most conspicuous and familiar Basidiomycota are those that produce mushrooms, which are sexual reproductive structures. Basidiomycota have a huge impact on human affairs and ecosystem functioning. Many Basidiomycota obtain nutrition by decaying dead organic matter, including wood and leaf litter. Thus, Basidiomycota play a significant role in the carbon cycle. Unfortunately, Basidiomycota frequently attack the wood in buildings and other structures, which has negative economic consequences for humans. Humans have found diverse uses for Basidiomycota. Mushrooms, both cultivated and wild, are eaten in many countries. For the untrained, mushroom-hunting is a risky endeavor, because some Basidiomycota produce deadly toxins.

Discussion:
From this exercise, I get a rough idea of Neurospora sexual reproduction process. I learned the technique for mating and picking perithecium. This exercise teaches me some fundamental techniques to work with ascomycetes.

By observing morphology of different mushroom, I learn mushrooms belong to Basidiomycota phylum. The knowledge from textbook can correlate to our daily life. We should not taste wild mushroom if we are not 100% sure about the safty since many wild mushrooms produce toxin.

Lab 7 Fluoresence microscopy and protein localization; continuation of Neurospora genetics

What's learned
1.Fungal genetic stock center website (www. fgsc. net). The stock center contains all experimental fungal strains. Knockout lines of certain genes can be ordered from the stock center. For example,it provides all information for Neurospora.

2. Knock out a fungal gene: homologous recombination.
This method can directly replace the coding region to block the gene function completely. It has advantage over point mutation.

Objective:
1.Pick single ascospore from mating plate for heat shock.

2.Observe nuclei, actin, tubulin, Mak-2 under fluorescence microspores.

3.Observe mushroom/Chlorophylum sp. basidiospores, Buller's Drop and hyphae clamp connection.
The purpose for this exercise is to practice on picking single ascospores from agar block, and taking video for Neurospora hyphae growth. Additionally,to get a general idea of some subcellular structure localization.

Material and Method:
Material:4% Water agar plate;picker,scaple,slides
Method:
1.Picking random ascospores

• Use scaple to cut water agar plate into 4cm block and put it on slide
• Spread loopful of spores-not too many because we want it well seperate
• Flame picker briefly-the advantage of the picker is it can cool down immediately.
• Use the picker as scaple to cut out a block with spores
• Pick up block of agar by scoping`0.2-0.3 mm square
• Transfer to agar tubes
• Transfer rack of tubes to refrigerator to allow spores to hydrate.
• Heat shock:Next day incubate the rack of tubes at 65 degree water bath for 45min.

2. Observer fluorescence signal from Nuerospora mutant.

• Cut a block of agar which is a little smaller than slide with hyphae tip in the middle. Gently lay the agar block on the slide and lay a long cover slide on the top without push. 
• Observe fluorescence signal under microscope. 

Result:
1. 10 ascospores are picked from the water agar block and transfer to slide medium agar tubes. Tubes are incubated at room temperature.

2. Observe microtubel tagged with GFP in SMRP11 mutant and nuclei tagged with GFP in SMRP10 mutant.
Under microscope I observe hyphae grow every second. The spizenkoper is wandering
because the hyphae is looking for nutrient source. Time lap is setup to capture each second of hyphae growth.

3. Mushroom/Chlorophylum sp. morphology observation:
Cut gill and observe basidispores.

4. Cut carp to observe hyphae and clamp connection.

Discussion:

From this experiment, I learned how to recognize ascospores and how to use a picker to pick every single ascospore. I also observe basidiospores and clamp connection from mushrooms. The clamp connection observation give me a vivid image of what clamp connection look like in natural condition which help me to understand the lecture better. I think we should do this kind of experiment more. After a lecture of some typical fungal morphogenesis, we are provided a chance to observe what it actually look like other than just get impression from textbook. It will help us to understand the lecture better and leave a longer time memory on the knowledge. 

Lab 5 Mitosporic fungi survey

Objective:
Observe conidia morphology of different fungi.
Method:
Regular mounting technique
Results:
1. Botrytis cinera: conidia are borne in grape-like clusters. Conidia are hyaline, ellipsoid to obovoid. There is contamination in Botrytis cinera. There are two conidia forms  present in the view. Based on my study, the small round ones are Botrytis cinera conidia.

2. Curvularia sp.: The poroconidia are curved slightly to distinctly, transversely septate, with an expanded third cell from the pore end of the conidium.

 

3. Epicoccum: Conidia look round, non-septate, brown to black in color, with both transverse and oblique septa, which makes them resemble a soccer ball.

4. Monilinia Fructicola: Conidia are hyaline (colorless), lemon-shaped, and produced in a moniloid manner (resembling a string of beads with constricted ends).

5. Rhizoctonia Solani: it does not produce spores and is hence identified only from mycelial characteristics or DNA analysis. Its hyphal cells are multinucleated. The hyphae are 4–15 μm wide and tend to branch at right angles. A septum near each hyphal branch and a slight constriction at the branch.

6. Thielaviopsis Basicola:  two types of conidia:

Type 1 conidial chains: terminal, with dark grey walls, smooth, in chains of (2)3-9 so appearing multiseptate, shortly cylindric with flat ends.

Type 2 phialoconidia: hyaline, smooth, cylindric, flat ended, very variable in size with 3-7 septate towards base.



lab 4 Dimorphism and mating: Zygomycete and Basidiomycete

Objective:
Observe the dimorphism for Mucor rouxii; observe ascospore, macroconidia and microconida for Neurospora; make cross for different mating type of Neurospora; inoculate Ustilago to maize seedlings and observe symptom development.
Method:
1. Different conidia form observation: use regular mounting method to observe dimorphism for Mucor rouxii; ascospore, macroconidia and microconida for Neurospora.
2. Cross: wet steriled loop, touch the side of tubes where Neurospora perithecia shot ascospores; inoculate the different mating type strain on opposite side of medium; incubate at room temperature for mating.
3. Ustilago infection:inject diluted  Ustilago spore suspension to maize stem.
Result:
1. Mucor rouxii has dimorphism, it can grow as yeast cell and hyphae. Dimorphism can be observed with a cross section Mucor culture medium. Hyphae will be observed on the upper layer. Yeast cell can be observed on the bottom layer. There are some transition cells in the middle layer.
Hyphae:

Budding yeast cell.

2. Conidiaphore of Neurospora mutant: microconidia don't break from each other.

Ascospore of Neurospora

Macroconidia and microconidia of Neurospora

Macroconidia develop under normal condition, microconidia develop under poor nutrition condition, mostly involved in mating. 

Neurospora mating: different mating type strain hyphae grow toward each other, fuse to produce perithecia along the lines hyphae meet. 

3. Ustilago inoculation: one week after inoculation, there are galls appeared on the leaves.

Galls will develop into lesions with times.