Food microbiology LAB exam

Know lab safety procedures

• label things, date, initials, sample

• nothing by mouth, in mouth

• wash hands before and after experiment

• decontaminate work area with disinfectant before and after experiment

• wear gloves, long pants, closed toed shoes, lab coat,

• dispose of everything in the correct are

• backpacks and all personal items in the back, water bottles and food outside of lab

• bunsen burner always attended, replace caps, inoculate loops

• treat all microbes as potentially harmful

• report spill and injuries to instructor

what PPE do we use in the lab?

• long sleeve lab coat, gloves, goggles

what is the purpose of immersion oil and when is it used?

• has same refraction as light which allows for more light through and allows for better resolution

• only used for 100x objective

how do you calculate the total magnification?

- 10 X objective = total magnification

Definition of a pure culture

1 microorganism only

Definition of the aseptic technique and why it is important in microbiology.

- Definition: series of steps and methods that reduce the introduction of contamination into samples collected or cultures being worked with

- Why important

- helps protect lab worker from the culture they are working with and potentially harmful effects

- its important to have clear results

Components (nutrients) in enriched media (agar plates, broth, etc.). Media can be solid or liquid.

- water

- carbs

- proteins

- lipids- not common but can serve as energy source

- trace minerals

- vitamins - not usually required

- Media can be

- Solid or liquid

What is turbidity and what does it indicate?

• What is it: cloudiness

• Indicates what: microbial growth

What is the streak plate method and why is it important? (ISOLATION)

- What is it: It is a dilution method that allows bacterial cells to be widely separated on agar so that as the single cell divides it produces a visible mass (colony).

- make 4 quadrants

- Why: to be able to observe the single bacterial cells → their morphology like shape, margin or edge, elevation, odor, and color

What dye is used for simple stains?

methylene blue

Gram stain: Know the reagents and what they do. (ORDER)

- 4 stains

- Primary stain: crystal violet (stains)

- Mordant: iodine (helps crystal violet stain be resistant to decolorizer)

- Decolorizer: acetone (removed crystal violet)

- Secondary stain: safranin

Know the color of gram positive and gram negative bacteria after Gram stain.

- Gram positive: violet

- Gram negative: pink/red

Endospores- what makes them unique and require heat for staining.

- They have thick outer coating called emporium

- Cryptobiotic state

- Require heat

What color do vegetative cells and endospores stain?

- Vegetative cells: pink (safranin)

- Endospores: green (malachite green)

Know how to kill endospores.

1. Autoclaving:

• High-pressure steam at 121°C (250°F) for at least 15–20 minutes.

2. Chemical Methods:

• Strong disinfectants like glutaraldehyde or ethylene oxide gas.

3. Incineration:

• Complete burning of contaminated materials.

4. Radiation:

• High-energy ionizing radiation can destroy spores

What stain is needed for endospore stain/what primary stain did we use?

• Primary Stain: Malachite green.

• Counterstain: Safranin.

  Be able to calculate colony-forming units.

CFU = number of colonies x 1/dilution factor x 1/volume plated

Know the definition of selective, differential, and enriched media

- Selective- inhibit growth of some bacteria and allow growth of bacteria of interest

- Differential - to distinguish between species by using chemical indicators that produce observable differences between different bacteria

- Enriched- formulates with all necessary ingredients for a wide variety of organism and grows multitude of bacterial species

Know the three different types of media we used in lab 6 and what bacteria each of those types selects/differentiates for.

- EMB(eosin methylene blue)

- Selects for Gram negative bacteria

- Differentiates for gram neg that ferment lactose

- What color does e coli turn → GREEEEEN metallic sheen

- MSA (mannitol salt media)

- Selects for staphylococcus aureus

- Differentiates for ability to ferment

- MacConkey

- Selective for gram negative bacteria

- Differential for ability to ferment lactose

Example of enrichment media

- Brilliant green agar- enriched media

EMB(eosin methylene blue)

- Selects for Gram negative bacteria

- Differentiates for gram neg that ferment lactose & sucrose; produces lactic acid

- What color does e coli turn → GREEEEEN metallic sheen; any other bacteria will appear pink to purple to dark brown

MSA (mannitol salt media)

- Selects for staphylococcus aureus

- Differentiates for ability to ferment; fermented mannitol turns media yellow

MacConkey (MAC)

- Selective for gram negative bacteria; contains bile salts & crystal violet

- Differential for ability to ferment lactose; bacteria that can ferment will form pink to red colonies

Phenol-red carbohydrate fermentation broths

Fermentation of glucose is indicated by a yellow tube further indicating the pH is low.

enrichment media

Brilliant green agar- enriched media

• Tryptic soy (TSA)

• Nutrient agar (NA)

• Blood agar

Know the importance of enzymatic systems in bacteria.

Drive microbial metabolism and growth; e.g., catalase, amylase.

What does catalase do?

- Responsible for the splitting of hydrogen peroxide into oxygen and water

What are the by-products of fermentable CHO?

- Lactic acid

- Acetic acid

- Liquids and gasses

• most bacteria’s produce acid **

What is sterilization? What methods can achieve sterilization?

- What is it: Complete destruction or elimination of all viable organisms in or on a substance being sterilized

- Methods: autoclaving, dry heat, very long boiling, incineration

- 2 methods that kill endospores:

- autoclaving and burning

What indicator organisms are used in the food industry? Why are they used?

- E.Coli, staphylococcus aureus, salmonella, acetobacter, lactococcus lactis.

- Why are they used?

- Assess either the microbiological quality or safety

What are facultative anaerobic organisms?

- Can grow with or without oxygen

What is food spoilage?

- Complex process involving a variety of organisms, food preservatives, and/or additives, food composition, temp, pH, and water activity

- Food is considered spoiled if sensory changes make it unacceptable to the consumer

Know the four factors that are associated with food spoilage discussed in Lab 9

- Color defects

- Texture changes

- Off smell or taste

- Slime

Know the definition of passive spoilage vs active spoilage

- Active spoilage - arises on healthy uncompromised products by plant pathogens

- Passive spoilage - wound induced from opportunistic organisms being able to gain entry into tissues that are damaged

- Bactericidal:

kills bacterial cells

- Bacteriostatic

inhibits the growth of bacterial cells (without killing them)

What methods can be used to control microbial growth?

- Temperature

- Aw

- pH

- Antimicrobial substances

- List 2 methods that can be used!!

1. Using disinfectants on inanimate objects

2. Taking antimicrobial drugs to kill microbes

• addition of sugars and salts lowers water activity

• Autoclave good at sterilizing; high pressure allows steam to reach higher temps than boiling; at 15 minutes can kill endospores **

Know the macroscopic fungi and microscopic fungi

- Macroscopic fungi: mushrooms

- Microscopic fungi: molds and yeast

Antiseptics do what?

They are microbicidal agents that can be applied to skin and mucous membranes

Disinfectants do what?

antimicrobial agents that act as cidal agents to varying degrees

Why is tartaric acid added into potato dextrose agar (PDA)?

- Lower pH to 3.5 to prevent spreading of colonies

- To reduce amount of spreading colonies

- Inhibit growth of bacteria but promote growth of fungi

What aw can be used to control the growth of most bacteria?

- <0.9

What is shelf-life?

- The time during which the food remains stable and retains its desired qualities

True or false: direct and indirect methods are used to count microorganisms

TRUE

What is when the slide is passed over burner 4-5 times

heat fixing

How can water be dinfected?

By boiling at 100 for 30 minutes

What spoilage organism is most responsible for baked goods spoilage?

• molds because they can

  1. Survive low water moisture activity

  2. Can tolerate acidic conditions

What organism is responsible for a wide variety of food spoilage and for soft rot of vegetables fruits meat fish milk?

Pseudomonas

What species is predominant in pasteurized milk?

Lactic acid bacteria