CFTRI M.Sc. (Food Technology) Microbiology Syllabus

What molecule makes Gram-negative bacteria's outer membrane endotoxic?

Lipopolysaccharide (LPS); its lipid A portion acts as the endotoxin.

What is peptidoglycan (murein) made of?

Repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by short peptide chains.

What is the purpose of staining microorganisms?

To increase contrast so cells (which are nearly transparent and colorless) are visible under the microscope, and to reveal shape, size, arrangement, and specific structures.

What is the difference between a simple stain and a differential stain?

A simple stain uses one dye to show overall shape/size/arrangement; a differential stain uses multiple reagents to distinguish between groups of cells or cell structures (e.g., Gram, acid-fast).

What is the difference between a basic dye and an acidic dye?

A basic dye has a positively charged chromophore that binds the negatively charged cell (direct/positive staining); an acidic dye is negatively charged and stains the background (negative staining).

List the four reagents of the Gram stain in correct order.

1) Crystal violet (primary stain), 2) Gram's iodine (mordant), 3) alcohol/acetone (decolorizer), 4) safranin (counterstain).

After a correct Gram stain, what color are Gram-positive and Gram-negative cells?

Gram-positive cells appear purple/violet; Gram-negative cells appear pink/red.

Why do Gram-positive bacteria retain crystal violet while Gram-negative do not?

The thick peptidoglycan of Gram-positives dehydrates and traps the crystal violet-iodine complex during decolorization; the thin wall plus lipid outer membrane of Gram-negatives lets the complex wash out, so they take up the counterstain.

What is the role of the mordant (iodine) in the Gram stain?

Iodine combines with crystal violet to form a larger, insoluble crystal violet-iodine (CV-I) complex that is harder to wash out of the cell.

What is the most critical and error-prone step of the Gram stain?

Decolorization with alcohol/acetone; over-decolorizing makes Gram-positives appear Gram-negative, and under-decolorizing makes Gram-negatives appear Gram-positive.

What does the acid-fast (Ziehl-Neelsen) stain detect and which organisms are acid-fast?

It detects cells with waxy mycolic-acid-rich walls; Mycobacterium and Nocardia are acid-fast (appear red); non-acid-fast cells appear blue from the methylene blue counterstain.

What is negative staining and what is it used for?

Staining the background with an acidic dye (e.g., nigrosin/India ink) while the cell stays unstained; used to visualize capsules and to view cells without heat-fixing distortion.

Which stain is used to demonstrate bacterial endospores, and what are its main reagents?

The Schaeffer-Fulton spore stain: malachite green (primary stain, driven in with heat) and safranin (counterstain); spores appear green, vegetative cells red/pink.

What is the purpose of heat fixation before simple/differential staining?

It kills and adheres cells to the slide, preserves morphology, and makes them more permeable to the stain (prevents washing away).

Define a food contaminant.

Any biological, chemical, or physical agent or foreign matter not intentionally added to food that may compromise its safety or quality.

Name the three main categories of food contamination hazards.

Biological (bacteria, viruses, molds, parasites), chemical (toxins, pesticides, heavy metals, cleaning agents), and physical (glass, metal, hair, stones).

What is cross-contamination and how is it prevented?

The transfer of harmful microbes from one food/surface to another (e.g., raw to cooked); prevented by separating raw and cooked foods, using separate utensils/boards, handwashing, and sanitizing surfaces.

Differentiate food infection from food intoxication.

Food infection results from ingesting live pathogens that grow in the body (e.g., Salmonella); food intoxication results from ingesting a preformed toxin in the food (e.g., Staphylococcus aureus, Clostridium botulinum).

What organism causes botulism and what conditions favor it?

Clostridium botulinum; it is anaerobic, spore-forming, and produces a potent neurotoxin in low-acid (pH > 4.6), low-oxygen canned/preserved foods.

What are the seven principles of the HACCP food safety system?

1) Conduct hazard analysis, 2) Determine critical control points (CCPs), 3) Establish critical limits, 4) Establish monitoring, 5) Establish corrective actions, 6) Establish verification, 7) Establish record-keeping/documentation.

What is the 'temperature danger zone' for bacterial growth in food?

Approximately 5 degrees C to 60 degrees C (about 40-140 degrees F), the range in which pathogenic bacteria multiply most rapidly.

What is water activity (aw) and why does it matter for food safety?

Water activity is the ratio of the vapor pressure of water in food to that of pure water (0-1); lowering aw inhibits microbial growth. Most bacteria need aw above ~0.91, most molds above ~0.80.

What are the main objectives/principles of food preservation?

To prevent or delay microbial decomposition, prevent self-decomposition (enzyme/autolysis) and chemical changes (oxidation), and prevent damage from insects, pests, and physical/mechanical injury.

What is the difference between asepsis and the removal of microorganisms as preservation principles?

Asepsis means keeping out (preventing entry of) microorganisms, e.g., packaging; removal means physically taking microbes out, e.g., filtration, washing, centrifugation.

How does high temperature preserve food, and what is the difference between pasteurization and sterilization?

Heat destroys microbes and enzymes. Pasteurization is mild heat killing pathogens/vegetative cells (food not sterile, e.g., HTST milk 72 C/15 s); sterilization (e.g., canning/UHT) destroys all microbes including spores for commercial sterility.

How does low temperature preserve food, and what is the difference between chilling and freezing?

Low temperature slows microbial growth and enzyme activity (it preserves, not sterilizes). Chilling/refrigeration (0-5 C) slows growth; freezing (below -18 C) stops growth by removing available water as ice.

How does drying/dehydration preserve food?

It lowers water activity below the level needed for microbial growth and enzyme action, inhibiting bacteria, yeasts, and molds.

How do salt and sugar act as chemical preservatives?

High concentrations of salt or sugar lower water activity and create osmotic pressure that draws water out of microbial cells (plasmolysis), inhibiting their growth.

How does food irradiation preserve food and what are the dose-based categories?

Ionizing radiation (gamma rays, X-rays, electron beams) damages microbial DNA. Radurization (low dose, shelf-life extension), radicidation (medium dose, kills non-sporing pathogens), radappertization (high dose, commercial sterility).

What is the role of lowering pH (acidity) and fermentation in food preservation?

Low pH inhibits most spoilage and pathogenic microbes; fermentation by lactic acid bacteria produces acids (and sometimes alcohol/bacteriocins) that preserve food, as in curd, pickles, and sauerkraut.

What are the hurdle technology and the canning-relevant D-value and 12D concept in preservation?

Hurdle technology combines several mild preservation factors (pH, aw, temperature, preservatives) to inhibit microbes. The D-value is the time to reduce a population 10-fold at a set temperature; the 12D process (botulinum cook) reduces C. botulinum spores by 12 log cycles to ensure safe low-acid canned foods.