1. INTRODUCTION TO FOOD SAFETY AND QUALITY MANAGEMENT CURRICULUM (M
INTRODUCTION TO FOOD SAFETY AND QUALITY MANAGEMENT CURRICULUM (M. Phil) Program at FCC
Dr. Quratulain Syed
Chief Scientific Officer
Food & Biotechnology Research Centre, PCSIR, Labs, Complex ,Lahore

2. Course : Food Microbiology & Toxicology
Credit Hours: (2-1)
Objectives:
To understand different microbial threats related to food safety and epidemiology of different food borne illness.
To understand the international microbial limits for safe foods, toxicological aspect of foods and their impact

3. Food microbiology including microbial analysis, growth, physiology and survival;
Interpret the Codex & ICMSF microbiological criteria regulations for foodstuff;
Role and ability of antimicrobial agents and their role in cleaning science;
Comprehend results relating to the detection, enumeration, identification and prediction of micro-organisms; explain the etiology of common food borne illnesses/diseases;
Role of microbes in disease, food spoilage, food production,
Food preservation methods and biotechnology;

4. Importance of water quality, water chlorination (chemistry, methods, testing and interpretation of results) in food production.
Food toxicology: Overview - intrinsic and extraneous toxins; Principles, types, branches; Toxicity: curve, factors influencing potency, margin of safety, factors influencing toxicity; Dose-response relationship, manifestation of organ toxicity.
Measurement of toxicants and toxicity; Toxicokinetics: carcinogenesis, mutagenesis, teratogensis;

5. Practicals Safety in microbiological laboratory.
Basic functions and handling of laboratory equipments. Use of microscope.
Sterilization and disinfection of glassware.
Preparation of culture media. Staining of microorganisms and their structures.
Bacterial cultivation, growth measurement. Characteristics of bacterial colonies. Bacterial and fungal morphology. Micrometry.

6. 略
Microbial physiology 6

7. The Domain of Life
Comparative ribosomal RNA sequencing has defined the three domains of life:
Bacteria
Archaea
Eukarya

9. Prokaryotic Diversity
Several lineages are present in the domains Bacteria and Archaea
Enormous diversity of cell morphologies and physiologies

11. Cell Structure
Two structural types of cells are recognized: the prokaryote and the eukaryote.
Prokaryotic cells have a simpler internal structure than eukaryotic cells, lacking membrane-enclosed organelles.

14. Bacterial Morphology Some typical bacterial morphologies include
Coccus,
Rod / Bacilli
Coccobacilli
Spirillum,
Spirochete,
Appendaged,
Filamentous.

16. Cytoplasmic Membrane The cytoplasmic membrane
Highly selective permeability barrier
lipids and proteins
Forms a bilayer with hydrophilic exteriors and a hydrophobic interior.

19. Cell Wall
Gram-negative Bacteria have only a few layers of peptidoglycan , but gram-positive Bacteria have several layers.
In addition to peptidoglycan, gram-negative Bacteria contain an outer membrane consisting of lipo-polysaccharide (LPS), protein, and lipo-protein.

20. Cell wall Surrounds the cyptoplasmic membrane
Directly reflects adaptive strategies involved with
-Uptake of nutrients
- Excretion of waste products
-Movements
-Protection
-Adhesion
In some organisms >25 of the genome is devoted to its synthesis, regulation and maintenance

26. Gram positive cell wall:
Rigid structure
Based on a crossed-linked polymer
-Peptidoglycan
Also contains teichoic acids (2 types)
-Wall teichoic acids
Polymer consisting of ribitol and phosphate
Confer antigenic specificity for the bacteria

27. Gram negative membrane:
Consist of outer and inner (cytoplasmic) membrane separated by the periplasm
Outer membrane
- Flexible outer phospholipid bilayer with an inner
peptidoglycan layer
Strong negative charge of phospholipid bilayer helps evade phagocytosis
Also protects against some antibiotics
Outer membrane also contain hydrophobic lipopolysaccharides
and lipoproteins

28. Cytoplasmic (inner) membrane:
Periplasm:
- Solution between the inner and ouetr membrane
- Contains specific periplasmic proteins
Usually involved in hydrolysis and transport of materials
Cytoplasmic (inner) membrane:
Feature of both Gram-positive and Gram-negative cells
Phospholipid bilayer
Allow the pasage of membrane components through
Has peripheral or integral proteins associated with it

29. Chemically, bacteria consist of:
Water (75-85%) – bound water and free water
Dry matter (15-25%) – organic part and mineral substances (inorganic part)

30. Dry matter Organic part Inorganic part
proteins – 50-80%
nucleic acid – 10-30%
carbohydrates – 12-18%
polysaccharides – 3-5%
lipids – 5-10%.
Inorganic part
nitrogen (N), carbon (C), oxygen (O), hydrogen (H), phosphorus (P), sulfur (S), sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), iron (Fe) and other

31. Growth of Microbes Increase in number of cells, not cell size
One cell becomes colony of millions of cells 31

32. Growth of Microbes Control of growth is important for
Infection control
Growth of industrial products and biotech organisms 32

33. Cell Growth
Microbial growth involves an increase in the number of cells. Growth of most microorganisms occurs by the process of binary fission.
Alternative means
Budding
Conidiospores (filamentous bacteria)
Fragmentation

35. Generation Time Time required for cell to divide/for
population to double
Average for bacteria is 1-3 hours
E. coli generation time = 20 min
20 generations (7 hours), 1 cell becomes 1 million cells! 35

36. Fig. 7.14a 36

38. Growth and survival 38

39. Standard Growth Curve 39

40. Phases of Growth
Lag phase: adaptation to the environment making new enzymes in response to new medium
Exponential logarithmic growth: Desired for production of products . Most sensitive to drugs and radiation during this period
Stationary phase: nutrition exhausted, toxin increased
death rate = division rate
Decline: cell die (steady biomass) or lysis (decrease biomass) death exceeds division
Dormant as spore, non-viable state

41. Measuring Growth Direct methods – count individual cells
Indirect Methods – measure effects of bacterial growth 41

42. Measuring Microbial Growth
There are 4 surrogate ways:
Cell counting (direct),
Colony formation (direct) ,
Biomass determination (indirect),
Turbidity (indirect)
Microbes are useful tools in research because of their rapid life cycle, their simple growth requirements, and their small size.

43. Growth measurement
Cell count: microscopic observation; flow cytometer (direct)
Colony formation: Measure the living cell (direct)
Biomass determination: dry weight; essential cell component (indirect)
Turbidity (indirect)

44. 44

45. Counting the viable cells: Dilution
Agar : Solidifying medium

46. 46

47. 47

48. Indirect Counting
Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth. However, to relate a direct cell count to a turbidity value, a standard curve must first be established.

49. Turbidity 49

50. Metabolic Activity 50

51. Dry Weight 51

52. Factors Regulating Growth
Nutrients
Environmental conditions:
Generation time 52

53. Nutrients (Chemical Requirements)
Water
Elements
C (50% of cell’s dry weight)
Trace elements
Organic
Source of energy (glucose)
Vitamins (coenzymes)
Some amino acids, purines and pyrimidines 53

54. Water
Microbes require water to dissolve enzymes and nutrients required in metabolism
Water is important reactant in many metabolic reactions
Most cells die in absence of water
Some have cell walls that retain water
Endospores and cysts cease most metabolic activity in a dry environment for years
Two physical effects of water
Osmotic pressure
Hydrostatic pressure

55. Osmotic Pressure
Is the pressure exerted on a semi-permeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution
Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst

56. Osmotic Pressure
Hypertonic solutions have greater solute concentrations; cells placed in these solutions will undergo cremation (shriveling of cytoplasm)
This effect helps preserve some foods
Restricts organisms to certain environments
Obligate halophiles – grow in up to 30% salt
Facultative halophiles – can tolerate high salt concentrations

57. Hydrostatic Pressure Water exerts pressure in proportion to its depth
For every addition of depth, water pressure increases 1 atm
Organisms that live under extreme pressure are barophiles
Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape

58. Environmental Effects on Microbial Growth

59. Factors affecting Growth
The orderly increase in the sum of all the components of an organism
Affected by:
Nutrients
pH: neutrophils, acidophils, alkalophils
Temperature: psychrophils; mesophils; themophils, thermodurics
Aeration
Pressure
Ionic strength and osmotic pressure: halophils, osmophils

60. 1. Temperature Temperature is a major environmental factor
controlling microbial growth.
The cardinal temperatures
- minimum, optimum, and maximum.

62. Microorganisms can be grouped by the temperature ranges they require.

63. 2. Low or High pH The acidity or alkalinity of an environment
can greatly affect microbial growth.
Organisms that grow best at low pH are
called acidophiles; those that grow best at
high pH are called alkaliphiles.

65. or high pH, but most organisms grow best between pH 6 and 8.
Some organisms have evolved to grow best at low
or high pH, but most organisms grow best between
pH 6 and 8.
The internal pH of a cell must stay relatively close
to neutral even though the external pH is highly
acidic or basic.
Acid (below pH 4) good preservative for pickles,
cheese

66. pH
Many bacteria and viruses survive low pH of stomach to infect intestines
Helicobacter pylori lives in stomach under mucus layer 66

67. 略
3. Air requirement:O2
Aerobe: A microorganism whose growth requires the presence of air or free oxygen
Anaerobe: A microorganism that grows only or best in the absence of free oxygen. Organisms utilize bound oxygen
Microaerophile: A microorganism that grows best in the presence of low concentrations of oxygen

68. Facultative anaerobe/aerobe: A microbe that adjusts its metabolism to depending on the oxygen concentration in which it is growing. can live with or without oxygen.
Aerotolerant anaerobe: an organism that always grows in an anaerobic mode -- can tolerate oxygen and grow in its presence even though they cannot use it.
Capneic microbe: An organism that requires 3 to 10% CO2 for growth

70. Growth pattern Obligate aerobe Obligate anaerobe Microaerophile
Aerotolerant anaerobe
Facultative anaerobe/aerobe 70

71. Two types of biofilms when cells grow as biofilm
Environmental
Disease-associated
Symbioses
Termite, ruminant digestion
Sewage treatment bioreactors
Water pipes
Dental units
Contact lens cases
Dental plaque
Endocarditis
Cystic Fibrosis
Otitis media
Urinary catheter
Implants

72. Special techniques are needed to grow aerobic and anaerobic microorganisms.

73. Several toxic forms of oxygen can be formed in the cell as the result of respiration, but enzymes are present that can neutralize most of them. Hydrogen peroxide is one of those forms that can be neutralized by catalase.

74. 4. Salinity
Some microorganisms (halophiles) have evolved to grow best at reduced water potential, and some (extreme halophiles) even require high levels of salts for growth.
extreme halophiles can live in solutions of 25 % salt; seawater = 2% salt

75. 75

76. Extremophiles Definition - Lover of extremities Temperature extremes
Boiling or freezing, 1000C to -10C
Chemical extremes
Vinegar or ammonia (<5 pH or >9 pH)
Highly saline, up to x10 sea water
we sterilize & preserve foods today

77. Extreme Temperatures Thermophiles - High temperature
Thermal vents and hot springs
May go hand in hand with chemical extremes
Psychrophiles - Low temperature
Arctic and Antarctic
1/2 of earth’s surface is oceans between 1-40C
Deep sea –10C to 40C
Most rely on photosynthesis

78. Chemical Extremes Acidophiles - Acidic
Again some thermal vents & hot springs
Alkaliphiles - Alkaline
Soda lakes in Africa and Western U.S.
Halophiles - Highly saline
Natural salt lakes and manmade pools

79. Extremozymes Enzyme from Extremophile Industry & Medicine
What if you want an enzyme to work
In a hot factory?
Tank of cold solution?
Acidic pond?
Sewage (ammonia)?
Highly saline solution?
Pay a genetic engineer to design a “super” enzymes...
Extremophiles have the enzymes that work in extreme conditions

80. “Compatible Solute” Strategy
Cells maintain low concentrations of salt in their cytoplasm by balancing osmotic potential with organic, compatible solutes.
They do this by the synthesis or uptake of compatible solutes- glycerol, sugars and their derivatives, amino acids and their derivatives & quaternary amines such as glycine betaine.
Energetically synthesizing solutes is an expensive process.
Autotrophs use between 30 to 90 molecules of ATP to synthesize one molecule of compatible solute.
Heterotrophs use between 23 to 79 ATP.

81. Osmoregulation
Halophiles have adapted to life at high salinity in many different ways.
Structural modification of external cell walls- posses negatively charged proteins on the outside which bind to positively charged sodium ions in their external environments & stabilizes the cell wall break down.

82. Halophiles
The “salt-in” strategy uses less energy but requires intracellular adaptations. Only a few prokaryotes use it.
All other halophiles use the “compatible solute” strategy that is energy expensive but does not require special adaptations.

83. Mean Generation Time and Growth Rate
The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes.
Growth rate (min-1) =
Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

84. Classification of bacteria based on nutritional requirements
Autotrophs are free-living, most of which can use carbon dioxide as their carbon source. The energy can be obtained from:
sunlight – protoautotrophs (get energy from photochemical reactions)
inorganic compounds, by oxidation – chemoautotrophs (get energy from chemical reactions)
Heterotrophs are generally parasitic bacteria, requiring more complex organic compounds than carbon dioxide, e.g. sugars, as their source of carbon and energy.

85. Methods of laboratory diagnosis
Bacterioscopical (Microscopic examination)
Bacteriological (Culture method)
Detection sensitivity of bacteria to antibiotics
Serological
Biological
DNA-technology test (PCR)

86. In the clinical laboratory it is necessary:
isolate bacteria in pure culture;
obtain sufficient growth of bacteria for demonstration their properties such as study of morphological, cultural, biochemical, antigenic and pathogenic properties, bacteriophage and bacteriocin susceptibility;
determine a sensitivity
to antibiotics.

87. Methods of the cultivation
Streak culture (surface plating). The inoculum is spreaded thinly over the plate of a culture media in series of parallel lines in different segment of the plate. On inoculation well separated colonies are obtained over the final series of streaks.

89. Methods of the cultivation
Lawn or carpet culture. Lawn cultures are prepared by flooding the surface or plate with suspension of bacteria. It provides uniform surface growth of bacteria. It is useful for bacteriophage typing and antibiotic sensitivity test.

90. Methods of the cultivation
Stroke culture. It is made in tubes containing agar slopes. It is used for providing a pure growth of bacterium (for slide agglutination).

91. Methods of the cultivation
Stab culture. It is prepared by puncturing with charged long straight wire (loop). Stab culture is employed mainly for cultivation of anaerobes.

92. Pure plate culture

93. Methods of the cultivation
Liquid culture in a tube, bottle or flask may be inoculated by touching with a charged loop

94. Identification of bacteria
Microscopic examination: It helps to detect a shape, a size and an arrangement of microorganisms
Staining reaction: On gram staining we can have two groups of microorganisms: Gram positive and Gram negative.
E. coli, Gram negative (A), Staphylococcus epidermidis,
Gram positive (B) and Bacillus cereus, Gram positive

95. Identification of bacteria
Motility: Some bacteria can move (Salmonella, E. coli, Proteus, Pseudomonas, Vibrions, Clostridia). Dark ground microscopy and Phase contrast microscopy, special culture media use for studying motility of bacteria
Special stain for flagella

96. Identification of bacteria
Culture character: Growth requirement, colonial characteristics in culture
Colony morphology descriptions

97. Colony morphology

98. Identification of bacteria
Metabolism: Capacity to form pigment and power of haemolysis is help for classification of bacteria
Staphylococcus
aureus
Micrococcus
roseus
Studying of
haemolysis

99. Colonies and pigments of bacteria

100. Identification of bacteria
Biochemical reactions: The more important and widely used tests are as under:
a) Sugar fermentation

101. Identification of bacteria
b) Indole production
c) Hydrogen sulfide production

102. Identification of bacteria
d) Other tests: Citrate utilization; Nitrate reduction; Methyl red test; Urease test; Catalase test; Oxidase reactions.
Positive Catalase Test
on Staphylococcus aureus
Negative Catalase Test
on Streptococcus lactis

103. API-20 "Bio Merieux" (France) strip test
Twenty tests are performed on this strip by a simple procedure, saving time and money.
Escherichia coli
Enterobacter agglomerans
Edwardsiella hoshinae

104. Identification of bacteria
Antigenic analysis: by using specific sera we can identify microorganism by agglutination reaction (Serologic Typing of Shigella).
The clumping of the bacteria
is seen in this circle
No clumping of the bacteria
is seen in this circle

105. Identification of bacteria
Pathogenicity: For pathogenicity test commonly used laboratory animal models are guinea pig, rabbit, rat and mouse.
Resistance to antibiotics and other agents

106. Metabolism is the process of building up chemical compounds in the cell and their breaking down during activity to receive the required energy and the building elements.
Metabolism comprises of anabolism (assimilation) and catabolism (dissimilation)

107. Classification of Media
A. On the basic of consistency:
Solid media
Liquid media
Semisolid media

108. Classification of Media
Nutrient media can be subdivided:
1. Simple media - meat-peptone broth (MPB), meat-peptone agar (MPA)
2. Synthetic media
3. Complex media
4. Special media: a) Enriched media; b) Enrichment media; c) Selective media; d) Indicator and differential media; e) Sugar media; f) Transport media.

109. 5. Aerobic and anaerobic media - according to type of respiration bacteria subdivided into 4 groups:
Obligate aerobes (Brucella)
Microaerophils (H.pylori)
Obligate Anaerobes (C.tetani)
Facultative Anaerobes (E.coli)

110. Autoclave (steam under pressure)
Sterilization
TREAT-MENT
TEMPE-RATURE
EFFECTIVENESS
Incineration
>500 C
Vaporizes organic material on nonflammable surfaces but may destroy many substances in the process.
Boiling
100 C
Thirty minutes of boiling kills vegetative forms of bacteria but may not kill bacterial endospores. There are also toxins that are not inactivated at 100C.
Intermittent boiling
Three 30-minute intervals of boiling, followed by periods of cooling kills bacterial endospores.
Autoclave (steam under pressure)
121 C for 15 minutes at 15 p.s.i.
Kills all forms of life including bacterial endospores. The substance being sterilized must be maintained at the effective temperature for the entire time.

111. Pasteurization (batch method) Pasteurization (flash method)
Sterilization
Dry heat (hot air oven)
160 C for 2 hours
Used for materials that must remain dry. Good for glassware, metal, but not most plastic or rubber items.
170 C for 1 hour
Same as above. Note that increasing the temperature by 10 C shortens the sterilizing time by 50 %.
Pasteurization (batch method)
63-66 C for 30 minutes
Kills most vegetative bacterial cells, including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis.
Pasteurization (flash method)
72 C for 15 seconds
Effect on bacterial cells is similar to batch method. For milk, this method has fewer undesirable effects on quality or taste.

112. AUTOCLAVES (1) AND HOT AIR OVEN (2)