# Lab 7 Yeasts and Molds

## BACKGROUND

Fungi are eukaryotic organisms, distinct from bacteria due to their complex cellular structures, including true nuclei, endoplasmic reticulum, and mitochondria. Fungi's reproductive capabilities, both sexual and asexual, combined with their metabolic processes, form the basis of their classification, primarily based on morphology.

Molds, a type of fungi, grow as intertwined filaments called hyphae, collectively known as mycelium. Hyphae are categorized into vegetative, which provide nutrients, and fertile, which serve as reproductive structures. A key classification feature is whether hyphae are septate (divided into cells by crosswalls) or non-septate (multi-nucleate cells without crosswalls).

Yeasts differ from molds in that they usually don't form hyphae and reproduce through budding or fission. Fungi, including yeasts and molds, can contribute positively to food production, such as in wine fermentation, or negatively by producing toxins, posing a safety concern. They can survive or thrive in conditions that are challenging for bacteria, such as environments with low pH or high salt/sugar levels.

### Yeasts:

- ***Saccharomyces cerevisiae***: Utilized in the production of beer, wine, and baked goods. Known for its use in molecular and cell biology studies, it reproduces by budding and is also used in nutritional yeast products.
- **Kluyveromyces fragilis**: Found in spoiled milk products, this yeast is the sexual stage of Candida pseudotropicalis. It varies in color and size and is notable for its pigmentation.
- ***Candida utilis***: Employed as a food additive for flavoring and in biotechnology for heterologous gene expression. It is closely related to Cyberlindnera jadinii.
- **Yarrowia lipolytica**: Known for its ability to utilize unusual carbon sources, including hydrocarbons, and recognized as a safe novel food by the European Food Safety Authority.
- ***Zygosaccharomyces rouxii***: An osmophilic yeast involved in the spoilage of high-sugar content foods and used in the fermentation of soybeans for soy sauce and miso.
- ***Debaryomyces hansenii***: An osmo-, halo-, and xero-tolerant yeast that can produce toxins and is commonly found in various types of cheese.

### Molds:

- ***Mucor racemosus***: Exhibits both filamentous and yeast-like morphologies, used in the production of certain oriental foods and involved in food spoilage.
- ***Rhizopus nigricans***: Causes spoilage in various foods and is used in the production of tempeh and alcoholic beverages.
- ***Aspergillus niger***: Known as "black mold" on fruits, used in the commercial production of acids, and capable of causing food spoilage.
- ***Penicillium chrysogenum***: Produces the antibiotic penicillin and can cause soft rot in fruits.
- ***Geotrichum candidum***: A yeast-like fungi causing post-harvest rot of fruits and vegetables, often found in food processing environments.

## OBJECTIVES

- Practice mold and yeast isolation techniques.
- Familiarize with the microscopic and macroscopic morphology of fungi.

## MATERIALS

### Cultures:
- 6 known and two unknown yeasts
- 1 sporulating yeast culture
- 5 known and 2 unknown molds

### Food sample:
- Fruit juice

### Media:
- Standard methods agar (SMA) = Plate count agar (PCA)
- Acidified potato dextrose agar (PDA-acid) plates
- Yeast extract-malt extract (YM) plates
- 0.1% peptone water for making dilutions (4.5 ml tubes)

### Yeast identification media:
- Yeast Carbon Base (YCB) with KNO3 as nitrogen source
- Yeast Nitrogen Base (YNB) plates with various 1% carbon sources (e.g., glucose, erythritol, lactose, sucrose, glycerol, galactose, no sugar)
- Yeast extract plates containing 50% glucose and a combination of 5% Glucose and 12% NaCl

### Supplies:
- Sterile applicator sticks
- Patch pattern for plate organization
- Peptone water dilution blanks
- Mold/yeast teaching slides for reference

## PROCEDURES
### Procedures [VP for lab 7](https://drive.google.com/file/d/1SztOiEoke-ikL6MXvhq9jSdRL4y5a7I8/view?usp=drive_link)
### STEP 1: Isolation from Food Samples
- Serially dilute the fruit juice sample and plate on SMA and PDA-acid to isolate fungi. Incubate at room temperature and later record counts for bacteria, yeasts, and molds.

### STEP 2: Identification of Yeast Unknowns
- Microscopically examine known and unknown yeasts, documenting colony morphology. Incubate

cultures on YM plates at 30°C and describe the colony morphology post-incubation.

### STEP 3: Growth on Various Media
- Inoculate yeast cultures onto different nutrient and stress condition plates to assess growth, osmophilic, and salt tolerance characteristics.

### STEP 4: Sporulating Yeast Cultures
- Examine and sketch the sporulating yeast culture, focusing on the ascus and number of ascospores.

### STEP 5: Observation of Molds
- Using a stereo microscope, examine and sketch the mold cultures, noting structures associated with asexual spore production and hyphal arrangement.

### STEP 6: Identification of Unknown Molds
- Microscopically and macroscopically examine the unknown mold cultures to identify based on observed characteristics.

## DISCUSSION QUESTIONS

- Discuss the challenges in differentiating mold/yeast morphologies and propose solutions to improve identification processes.

This lab guide provides a comprehensive overview of the objectives, materials, and procedures for studying yeasts and molds, emphasizing the importance of fungi in food safety and production.