BP303T PHARMACEUTICAL MICROBIOLOGY

WELCOME
To
SEM-III
 BP303T PHARMACEUTICAL MICROBIOLOGY

Course Objectives:
Upon completion of the subject student shall be able to;
1. Understand methods of identification, cultivation and
preservation of various microorganisms
2. To understand the importance and implementation of
sterilization in pharmaceutical processing and industry
3. Learn sterility testing of pharmaceutical products.
4. Carried out microbiological standardization of
Pharmaceuticals.
5. Understand the cell culture technology and its
applications in pharmaceutical industries.
 UNIT 1
❑ Introduction, History, Branches, Scope, Importance of microbiology

❑ Introduction to Prokaryotes and Eukaryotes

❑ Study of ultra-structure and morphological classification of bacteria

❑ Nutritional requirements & raw materials used for culture media

❑ Physical parameters for growth & growth curve

❑ Isolation and preservation methods for pure cultures, cultivation of anaerobes

❑ Quantitative measurement of bacterial growth (total & viable count).

❑ Study of different types of phase contrast microscopy, dark field microscopy and

electron microscopy.
fermentation
 PHARMACEUTICAL MICROBIOLOGY

Unit 1
a) Introduction to Prokaryotes
and Eukaryotes
b) Morphological Classification
of Bacteria
- By Ms. Christina V
a) Introduction to Prokaryotes and Eukaryotes 2
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PARTS OF BACTERIA/CELL COMPONENTS 5
(PROKARYOTIC CELL)
1. CAPSULE
2. CELL WALL
3. CELL MEMBRANE
4. CYTOPLASM
5. NUCLEOID
6. INCLUSION BODY
7. RIBOSOME
8. MESOSOME
9. FLAGELLA
10. PILLI
 CELL COMPONENTS 6
 CAPSULE
• Outer most thick structure which is rigid and flexible
• Composition (98% water + 2% glycoprotein)
• It is the identity of bacteria and its virulence
• It protects the bacteria and gives shape to it
• Enables the cell to attach to surfaces in its environment
➢ CELL WALL
• It is also a thick structure made up of peptidoglycan layers, sugars, and
amino acids
• We can identify bacteria as gram –ve / gram +ve based on their cell wall
• Gram +ve=20-80nm thickness of peptidoglycan
• Gram –ve=7-8nm thickness of peptidoglycan
• It also gives protection, shape and identification of bacteria
 CELL COMPONENTS 7
➢ CELL MEMBRANE
▪ It is a semi-permeable membrane
▪ It is a thin layer in prokaryotic cell made up of
phospholipid bilayer
➢ CYTOPLASM
▪ All the material inside the cell except nucleus
➢ NUCLEOID
▪ It is less developed nucleus, thus it is called as Nucleoid
▪ Nucleoid contains 60% DNA, 30% RNA, 10% Protein
➢ RIBOSOME
▪ It helps in protein synthesis
▪ In bacteria, 70S type of ribosome is present
 CELL COMPONENTS 8
 INCLUSION BODY
▪ They are storage vessels that stores food
 MESOSOME
▪ It is slightly inside the cell wall
▪ It helps in cellular respiration
 FLAGELLA
▪ It is a hair like structure that is 15-20 nm long
▪ It is used for locomotion
 PILLI/PILUS
▪ They are small hair like projections that exchange genetic material
during cell reproduction
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ASSIGNMENT-2

 DIFFERENCEBETWEEN :
a) PROKARYOTE & EUKARYOTE
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b) Morphological Classification of Bacteria
BASED ON MORPHOLOGY, BACTERIA CAN BE CLASSIFIED INTO 6 GROUPS
1. TRUE BACTERIA
2. ACTINOMYCETES
3. SPIROCHAETES
4. MYCOPLASMA
5. RICKETTSIAE
6. CHLAMYDIAE
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1. TRUE BACTERIA
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a) Cocci: Spherical/ oval
Eg: Micrococcus flavus

Eg: Diplococcus pnemoniae

Eg: Gaffkya tetragena

Eg: Streptococcus pyogenes

Eg: Sarcina ventriculi

Eg: Staphylococcus aureus

b) Bacilli-Rod shaped 17

Eg: Bacillus cereus

Eg: Moraxella bovis

Eg: Lactic acid bacilli

Eg: Streptobacillus moniliformis

Eg:?

Eg: Bacillus anthracis

2) ACTINOMYCETES 18

• They are gram +ve bacteria

• Mostly present in the soil
• The actinomycetes are an important source of antimicrobial
compounds, they produce up 60%-70 of the total antibiotics.
3) SPIROCHAETES 19

• They are gram -ve bacteria

• It has double membrane
• Eg: Treponema pallidum-Syphilis
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4) MYCOPLASMA
They are oval in shape and lack rigid cell wall
They are underdeveloped cells and contain only some organelles and
DNA (Eg: Mycoplasma genitalium-World’s Smallest Bacteria)
It causes diseases in Animals (Eg: Mycoplasma bovis-causes
bronchopneumonia, mastitis in cattle)

5) RICKETTSIAE
They look like filaments and have no branching and chains
It is non-motile
It is a gram –ve bacteria (Eg: Rickettsia rickettsia-Rocky Mountain
spotted fever-RMSF)

6) CHLAMYDIAE
They are very small, oval shaped and has
peptidoglycan+protein in it (Eg: Chlamydia pneumoniae-Pneumonia)
 PHARMACEUTICAL MICROBIOLOGY

Unit 1

a) Nutritional requirements
b) Raw materials used for culture media
c) Physical parameter for growth
d) Bacterial Growth Curve
- By Ms. Christina V
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a) Essential nutrients required by bacteria 3

 Bacteria requires energy and nutrient to build proteins and structural

membranes and derive various biochemical processes
 All bacteria obtain energy by oxidizing carbohydrates, lipids and proteins
 BASIC NUTRITIONAL REQUIREMENT
1. WATER:
 Major essential nutrient as it accounts for 80-90% of total weight of cell
 It is highly polar compound and it contains micronutrients and trace
elements required for the growth of bacteria
Essential elements- Major Macronutrients 4
 Macronutrients are the elements required by the bacteria in large amounts
 CARBON, HYDROGEN, OXYGEN, NITROGEN,PHOSPHOROUS, SULPHUR
2) CARBON:
 Carbohydrates is the principle source of carbon which is degraded by bacteria either
by oxidation or fermentation
 Autotrophic bacteria: They synthesize their own food
 Heterotrophic bacteria: Use organic compounds as carbon source
3) NITROGEN:
 It is the obtained from Ammonia
 It is the main source for synthesizing protein and nucleic acid for bacteria
Macronutrients 5

4) SULPHUR:
 Many bacterial species use sulphur from organic sulphur, inorganic
sulphur compounds and elemental sulphur.
 Sulphur helps in making several co-enzymes and to synthesize cysteinyl
and methionyl side chain of protein

5) PHOSPHOROUS:
It is required to synthesize nucleic acids, ATP, Coenzymes, flavins
Micro-nutrients 6

 Mineral sources
 These are K+, Ca++, Mg++, Fe++, Cu++, Cobalt, Manganese, Zn++,
required in trace amount
 It can be obtained via tap water or as contaminants from other medium
ingredients
 Source of energy
 CO2 is required by all bacteria and is important for cellular metabolism
 Energy source is based on nature of bacteria
 Aerobic bacteria: Require O2 for growth
 Anaerobic bacteria: Grows in absence of oxygen
 Growth factors 7
 These are organic compounds in small quantities
 Bacteria needs growth factors like pyrimidine and purine for the formation of DNA and
RNA
 Vitamins
 In many cases, growth factors are identical with Vitamins from B group like: Thiamine,
Riboflavin, Nicotinic acid, pyridoxin, folic acid and vitamin B12
b) RAW MATERIALS USED FOR CULTURE MEDIA 8
 CULTURE MEDIA
 Culture media is a gel or liquid that contains nutrients and is used to grow bacteria or
microorganisms. They are also termed growth media.
 Different cell types are grown in various types of medium. Nutrient broths and agar plates are
the most typical growth media for microorganisms.
 Some microorganisms or bacteria need special media for their growth.
 RAW MATERIALS: Materials used for making the culture media are:
 Water
 Peptones
 Meat extract
 Yeast Extract
 Agar
RAW MATERIALS USED FOR CULTURE MEDIA 9
1. WATER
 In bacteria, 70-80% of water is present and it is used as a vehicle
 Water is essential for preparation of culture/nutrient media
 Distilled water is always preferred to use
2. PEPTONE
 Partially digested proteins obtained from meat, fibrin, soya
 It is the NITROGEN source for bacteria
 It contains protease, amino acids, etc
 Acts as a buffer
 It is hygroscopic in nature
RAW MATERIALS USED FOR CULTURE MEDIA 10
3. Meat Extract
 It is prepared from fresh lean meat by hot water extraction process
 Contains gelatin, creatin, purine, mineral salts, carbohydrates etc

4. Yeast Extract
 It is prepared from cells of Baker’s yeast or Saccharomyces
 Contains carbohydrates, amino acids, growth factor
 Used as vitamin source
 5. Agar 11
 Golden yellow granular powder
 Obtained from different seaweeds algae (red algae)
 It has no nutritive value
 It is used for preparing the solid medium
 It has high resistance to all micro-organisms
 It gets solidified below 40°C
 It is easily available and economical
c) PHYSICAL PARAMETERS FOR GROWTH 12
 1. TEMPERATURE

Pseudomonas vibrio

[Link], Streptococcus pneumoniae

Thermus aquaticus
2. pH 13
3. Gaseous Requirement 14
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4. OSMOTIC PRESSURE
 Microbes grow well in isotonic environment
 But their growth decreases in hyper/hypo osmotic medium

5. LIGHT
 Bacteria are sensitive to light and other radiations
 So, majority bacteria grow in absence of light (darkness)
d) Bacterial Growth Curve 16
No. of bacteria vs Time (Bacterial Growth Curve) 17
BACTERIAL GROWTH CURVE 18
1. LAG PHASE: (1-4hrs)
 In this phase, there is no growth of bacteria because the culture media is
just prepared and therefore, upto 2-4hrs, there will be no growth of
bacteria.
 The bacteria is adapting itself to the growth conditions
2. LOG PHASE/EXPONENTIAL PHASE (8hrs)
 In this phase, there is maximum growth of bacteria
 They reproduce at a fast rate and number of bacteria increases
 This phase will eventually decline when the nutrient media starts
decreasing
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3. STATIONARY PHASE
 In this phase, growth of the bacteria stops due to lack of nutrient media
 Therefore, bacterial growth remains stationary

4. DECLINE PHASE/DEATH PHASE (Few hrs to days)

 In this phase bacteria starts dying due to absence of nutrient media for growth and
other toxic elements are formed
ISOLATION AND PRESERVATION 21

METHOD FOR PURE CULTURE

Common Isolation
techniques:
1. Streak plate method
2. Pour plate method
3. Spread plate method
STREAK PLATE METHOD 22
POUR PLATE METHOD 23
SPREAD PLATE METHOD 24
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SERIAL DILUTION METHOD 27
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PRESERVATION METHOD FOR PURE CULTURE
 SUB-CULTURING
 REFRIGERATION
 CRYOPRESERVATION
 PARAFFIN METHOD
 LYOPHILIZATION
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QUANTITATIVE MEASUREMENT OF BACTERIAL GROWTH 36
 TOTAL COUNT
 COUNTING CHAMBER METHOD
 ELECTRONIC COULTER COUNTER METHOD

 VIABLE COUNT
 PLATE COUNT METHOD
 MEMBRANE FILTER COUNT METHOD

 INDIRECT METHOD
 TURBIDIMETRIC METHOD
1. TOTAL COUNT METHOD 37
 COUNTING CHAMBER METHOD
 The most obvious way to count microbial numbers is through direct
counting.
 Petroff-hausser counting is one of the easiest and accurate way to
count bacteria using HAEMOCYTOMETER OR NEUBAUERS
SLIDE
 Side view of the chamber showing the cover glass and the space
beneath it that holds a bacterial suspension.
 A top view of the chamber. The grid is located in the center of the
slide.
 An enlarged view of the grid. The bacteria in several of the central
squares are counted.
 Concentration of the cells can be calculated by using the average no.
of bacteria in these squares.
 1 square=50-60 bacteria
 Eg: 12 squares=12 X 50=600 Bacteria
 ELECTRONIC COUNTER METHOD
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 In this method, Coulter Counter instrument is used for
measurement
 In this, a bacterial suspension is passed through the capillary
tube, and there is an orifice which has very small diameter.
 Through this small orifice, only one bacterial cell is passed at
a time.
 When bacteria is passed, a current is passed and a voltage is
generated and 1 peak is formed
 So each time the bacteria passes through the orifice, voltage is
generated and peaks are formed.
 This gives the total count of bacteria
2. VIABLE COUNT: In this measurement, we have 39
to count only those bacteria which are alive

 PLATE COUNT METHOD

 It measures the cell density and provides
information regarding only viable bacterial cells
 Here, the microbial culture media is diluted several
times (serial dilution)
 After the dilutions, we take 1 ml from each dilution
and apply it on the agar plates individually and then
incubated
 After incubation, the no. of colonies can be seen
and they are counted.
 MEMBRANE FILTER COUNT METHOD
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INDIRECT METHOD 41
 TURBIDITY METHOD
 This is one of the most practical way of monitoring bacterial
growth
 The instrument used to measure turbidity is Spectrophotometer
 In this spectrophotometer, beam of light is transmitted through
a bacterial suspension
 As bacterial number increases, less light will reach the detector
and it will be registered on the instrument scale as %
transmission
 The amount of light striking the light sensitive detector on the
spectrophotometer is inversely proportional to the number of
bacteria under standardized conditions
 Lesser the light is transmitted, more the bacteria is present in
the sample
 PHARMACEUTICAL MICROBIOLOGY

UNIT 1
STUDY OF DIFFERENT TYPES OF PHASE CONTRAST
MICROSCOPY, DARK FIELD MICROSCOPY AND
ELECTRON MICROSCOPY
-BY MS. CHRISTINA V

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 WHAT IS A MICROSCOPE?
MICROSCOPE IS AN INSTRUMENT DESIGNED TO VIEW MAGNIFIED IMAGES OF SMALL
OBJECTS OR MICRO-ORGANISMS THAT CANNOT BE SEEN WITH OUR NAKED EYES

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BRIGHT AND DARK FIELD MICROSCOPY

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PHASE CONTRAST MICROSCOPE

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light

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APPLICATIONS OF BRIGHT AND DARK FIELD MICROSCOPY

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APPLICATIONS OF PHASE CONTRAST MICROSCOPY

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SCANNING ELECTRON MICROSCOPE (SEM)

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SCANNING ELECTRON MICROSCOPE

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TRANSMISSION ELECTRON MICROSCOPE (TEM)

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TRANSMISSION ELECTRON MICROSCOPE

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LIMITATIONS OF ELECTRON MICROSCOPE

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APPLICATIONS OF ELECTRON MICROSCOPE

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 GIVE DIFFERENCE BETWEEN:-
1. DARK FIELD MICROSCOPE AND PHASE CONTRAST MICROSCOPE
2. SCANNING ELECTRON MICROSCOPE AND TRANSMISSION ELECTRON MICROSCOPE

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