Andhra Pradesh state Govt. APSCHE JNTUA JNTUK · 2020. 7. 15. · GPAT Online classes...
Transcript of Andhra Pradesh state Govt. APSCHE JNTUA JNTUK · 2020. 7. 15. · GPAT Online classes...
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GPATOnline classes
Pharmaceutical Microbiology and Biotechnology
(13th -16th June 2020)
Organized By
Andhra Pradesh state Govt. APSCHE JNTUA JNTUK
Presented ByMr.S.Hari hara sudhan . M.Pharm, MBA, (Ph.D)Department of Pharmaceutics,Raghavendra Institute of Pharmaceutical Education and Research (Autonomous)K.R.Palli Cross , ChiyyeduAnantapuramuAndhra Pradesh-515721
Day-2 (14-06-2020)
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RIPERAUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
GPATMicrobiology
• Syllabus
i. Introduction to Microbiology
ii. Microscopy and staining technique
iii. Biology of Microorganisms (Infections and causative organisms)
iv. Microbial spoilage
v. Vaccines & Sera
vi. Fungi and Viruses
vii. Aseptic Technique
viii. Sterilization & Disinfection
ix. Microbial Assay
Note: Red- Most Important topics
Green-Important topics
blue- lesser than red and green
Black-Go through once
All four days (13th to 16th June 2020) PPT contain Questions from the previous GPAT exams. (1998-2019). Students can practice the old questions after preparing the everyday's slide.
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Biology of Microorganisms Morphology and arrangements of Bacteria
StaphylococciStaphylococcus sp.
Divides in three plane
Cocci (Spherical Shape)
Micrococcus flavus.
Divide in one plane
Neisseria gonorrhoeae
Streptococcus pneumoniae,
Divides in one plane
Micrococcus sp.
Aerococcus, Pediococcus
and Tetragenococcus
Divides in two plane
Sarcina sp.
Divides in three plane
Streptococcus sp.
Divides in two plane
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Morphology and arrangements of Bacteria
Bacilli (Rod Shape)
Streptobacillus sp.
Corynebacterium sp.
Brucella sp.Bacillus cereus Coxiella burnetii
Curved/spiral
comma-shaped
Less than one turn
Or twist in the cell
Vibrio cholerae
rigid spiral structure.
many turns
typical bacterial flagella
Campylobacter jejuni,
Helicobacter pylori
Flexible spiral body.
many turns
Endoplasmic/periplasmic
flagella
Leptospira sp.
Treponema pallidum,
Borrelia recurrentis
Pleomorphic
No cellwall
No specific shape
Mycoplasma spFilamentous bacteria
Fungi like growth
Actinomyces
Note: Other Unusual shapes, square, star, Pear shaped, lobed spheres disks arranged like stacked coins, etc.
(Refer Microbiology by Pelczer page No.76) but no questions in past GPAT papers.
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RIPERAUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Ref:-Shahriar, Mohammad. (2013). Effect of Proteinase-k on DNA Extraction from Gram-positive Strains.
Structure of bacteria
Gram Negative
Presence of outer
membrane
surrounding thin
peptidoglycan layer.
Both are linked by
Braun’s protein
Rich in lipids.
endotoxins
Lipopolysaccharides
Lipid-A
O-Antigen
Presence of
periplasmic space.
Porins present
Less Peptidoglycan
Gram Positive
No outer membrane.
thick peptidoglycan layer.
Less amount of Lipid.
No periplasmic space
Teichoic Acid present
(combines with
magnesium ions and
protects from heat.)
High amount of
Peptidoglycan.
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of BacteriaFlagella (Motility)
1. Polar (at one or Both end of bacteria) (motility in liquid only)
Monotrichous (Single at one end)
(Pseudomonas aeruginosa)
Lophotrichous (Multi at one end)
(Pseudomonas fluorescens)
Amphitrichous (Either single or cluster at both ends)
(Aquaspirillum serpens)
2. Lateral (Surrounding the cell) (motility in liquid
only). Peritrichous (Salmonella typhi)3. Atrichoous (No
flagella) Non motile
4. Spirochetes: Periplasmic/endoplasmic flagella having crawling
or creeping motility over solid surfaces also.
Spiroplasma
motile even in
viscous fluid
though
atrichous
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of Flagella
Three parts
1. Basal Body--- attached with cell
layers
2. Hook--- rotating outside
3. Filament (made of Flagellin
protein
Basal body- S, M rings at
cytoplasmic membrane
P-ring and peptidoglycan layer
L-ring at Outer membrane
Source picture-
https://i1.wp.com/microbeonline.com/wp-
content/uploads/2013/04/Structure-of-the-
prokaryotic-flagellum.jpeg
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RIPERAUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Motility of Bacteria
Chemotaxis- Moving towards or away from a chemical compound
Positive Chemotaxis- Moving towards chemical
Negative Chemotaxis- Moving away from Chemical
Phototaxis- Positive phototaxis only. Photosynthetic bacteria towards light
Magnetotaxis- towards magnetic earth field with help of magnetosomes-type of
inclusion body
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of Bacteria
Pili- shorter than flagella, More in numbers than flagella, throughout cell surface, hollow.
Not for motility.
For pathogenecity, attachment over cell surface, sex pili formation to transfer plasmid
Capsule- if its thin –Microcapsule Thick and many cells are embeded-Slime
Made of Homopolysaccharides(synthesized outside by secreting enzyme outside and
breaking the carbohydrates)
Or
Made of Heteropolysaccharides (Synthesized inside the cell and transported outside and
capsule created
For protect against drying, prevent bacteriophages entry, escape from immunity and create
invasiveness for cell during pathogenecity, attachment over cell surface, electrical charge to prevent
aggregation of cell and kept in suspension form
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RIPERAUTONOMOUS
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NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of Bacteria
Sheaths- cells enclosed in hollow tube
Prosthecae/stalks- semi rigid extension of cell wall and cytoplasmic membrane.
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of Bacteria
Cell Wall:-
Peptidoglycan (Murein)-
N-acetylmuramic acid,
N-Acetylglucosamine,
L-alanine,
D-alanine,
D-glutamate,
deamino acid
(LL or meso-diaminopimelic acid, or L-lysine, or
L-ornithine, or L-diaminobutyric acid)
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of Bacteria
Gram Negative
Presence of outer membrane surrounding thin
peptidoglycan layer.
(Both are linked by Braun’s protein)
Rich in lipids. Lipopolysaccharides, Lipid-A
O-Antigen (endotoxins)
Presence of periplasmic space.
Porins present
Less Peptidoglycan
Not susceptible to Lysozyme enzyme.
Gram Positive
No outer membrane. thick peptidoglycan layer.
Less amount of Lipid. (Except mycobacterium
and corynebacterium)
No periplasmic space
Teichoic Acid present
(combines with magnesium ions and protects
from heat.)
High amount of Peptidoglycan.
Easily destroyed by Lysozyme enzyme
Archaeobacterial cell wall:- No peptidoglycan,
Composed of Proteins, glycoproteins or polysaccharides. Some contain Pseudomurein (look
like peptidoglycan but chemical composition differs
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Structure of BacteriaCytoplasmic membrane- Covers cytoplasm, made of Phospholipids,
Contains Integral protein, peripheral proteins,
Phospholipids—Eubacteriae-Ester linked straight (unbranched) chain fatty acids
Archaeobacteria-Ether linked branched fatty acids
Protoplast- Gram positive cell without cell wall
Spheroplast- Gram negative cell without cell wall
Mesosome/membrane Intrusions-
Central mesosome- Cell division and DNA replication, DNA segregation into daughter cells
Peripheral mesosome- Septum formation during cell division.
Cyto plasmic inclusions/Gas Vacuoles- PHB (poly hydroxy butyric acid) granules, Metachromatic
granules/Volutin granules, starch granule, sulfur granules.
Gas vacuole without any membrane
Nuclear material- DNA at Nucleoid region, No nuclear membrane, No histone, Plasmid in
cytoplasm
Ribosome- 70S
Spore- Endospore/Exospore, contains Dipicolinic Acid (DPA), Calcium- DPA provide heat
resistant
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Source of energy
Phototroph
(light)Chemotroph
(Chemicals)
Source of electron
Lithotroph
(Inorganic compounds)
Organotroph
(Organic compounds)
Photolithotroph
Photoorganotroph
Chemolithotroph
Chemoorganotroph
Source of Carbon
Autotroph
(Atmospheric CO2)
Heterotrophs
(organic or inorganic molecules)
Types of microbes based on
Nutrients
Obligate parasitic bacteria-Only live
intracellularlyMycobacterium
leprae, Rickettsias, chlamydias, spirocetes(Treponema Pallidum)
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RIPERAUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Types of microbes based on
temperature requirements
Psychrophiles
0℃- 20℃
Strict/obligate Psychrophiles
Die above 20℃
Faculative Psychrophiles
Optimum temperature
0℃
Can grow at 20℃-30℃
Mesophile
25℃- 40℃
Thermophiles
Grow best above 45℃
True Thermophiles/obligate
thermophiles/
stenothermophiles
Grows Only above 45℃
Faculative Thermophiles
Optimum temperature
45℃
Can grow at mesophilicrange also 25℃- 40℃
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RIPERAUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Types of microbes based on Gases
requirements
Aerobic
Require oxygen
Grows in top of liquid media (Pelicle type)
Anaerobic
Does not require oxygen, Oxygen in toxic
Grows in bottom of Media
(sediment type)
Nonstringent/Tolerentanaerobic
Can tolerate low level oxygen but wont use
for growth
Stringent/Strict/obligate Anaerobe
Cannot tolerate low level oxygen and dies
Faculative anaerobic
Can grow in presence and absence of oxygen.
Grows through out the media
(Suspended type)
Microaerophilic
Require low level of oxygen for growth
Grows below the surface of media
Note: Presence of superoxide dismutase, peroxidase, catalase provides oxygen tolerability to cells. Aerobic bacteria have these enzymes. But not anaerobic bacteria.
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Quantitative Measurement of Bacterial Growth
Cell count methods
Direct Microscopy
Coulter current
Colony count
Cell Mass methods
Turbidometry (spectrophotomenter0
Dry weight
Nitrogen content estimation
Cell activity
Biochemical activity
(Nutrient depletion rate of metabolite production rate)
Bacterial count methods
Total count methods
(Counting Dead and viable cells)
Direct microscopy
Counter current
Turbidometry
Dry weight
Nitrogen content estimatin
Viable Count methods
Counting only Live cells
Colony count
Biochemical Activity
Direct Count Methods
Direct microscopy
Counter current
Dry Weight
Nitrogen content estimation
Indirect count methods
Turbidometry
Biochemical Activity
Colony count
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RIPERAUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Sterilization
Complete removal of microbes and its spores1. Thermal death time:- Shortest period of time to kill a suspension of bacteria at a
prescribed temperature.
2. Decimal reduction time (D value)- Time required to reduce the microbial population by
one Log cycle (time to reduce the population by 90% )
Examples- A Culture containing 1 Lakh Clostridium botulinum cells
1. Thermal death time to kill all Clostridium botulinum at 100℃ is 300 minutes.
2. Thermal death time to kill all Clostridium botulinum at 110 ℃ is 35 minutes
3. Thermal death time to kill all Clostridium botulinum at 120 ℃ is 4 minutes
Decimal reduction time to kill 90% (one log cycle) of Clostridium botulinum at 70 ℃ is 20 hours (1 lakh reduced to 10000)-first cycle
second cycle kills another log cycle. Number will reduce to 10000 to 1000
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RIPERAUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Sterilization Methods (Physical)
Incineration- Direct heating over flame (hospital waste and Inoculation loop)
Mechanism: burning
Filtration- membrane filters (0.45 µm for bacteriological. 0.20 µm for viral)
Materials-Thermo liable materials
Heat sensitive Biological fluids
Mechanism: entrapment
Moist Heat- Autoclave oven
0 lb pressure = 100 ℃- 1 hour 5 lb pressure = 109℃- 40 Min10 lb pressure= 115 ℃- 30 Min15 lb pressure = 121 ℃ -15 Min20 lb pressure = 126 ℃ -3 MinMaterial- Liquids, plastics, Non hygroscopic
materials. Glassware with liquid media.Mechanism: Coagulation of proteins and
enzymes
Dry Heat- hot air oven160 ℃-2 hours180 ℃ -40 Min240℃ -10 Min320 ℃-3 secMaterial- Oily, powder, hygroscopic materials
Empty glassware(Can not use destructive material which can
not withstand high temperature)Mechanism: Oxidation of chemical
constituents
Radiation (cold sterilization)UV (2650 Å-Lethal) (surface sterilization)X-ray (costly) gamma rays (60Co )- Packed goodscathode rays-packed goodsMechanism:- UV-adjacent Thymine-
thymine dimer formation.Mechanism:- gamma , cathode, X-ray-
Electron removal from various molecules.
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Sterilization Methods (Chemical)
Gases:- (Closed Area/rooms/Material or equipments Kept in closed rooms)
Formaldehyde-Vapors of formaldehyde at room temperature of 22 ℃ humidity of 60 to 80%50-70mg/L concentration required
Surface sterilization only.
Mechanism:-Extremely reacts with Organic nitrogen compounds such as proteins and
nucleic acids.
Disadvantages-irritation to skin and eye, low penetrating power
Ethylene Oxide-
Vaporize at 10.8 ℃Highly flammable (mixed with Carbon dioxide or freon)
8.5% Ethylene oxide and 91.5% C02 require high pressure inside the the area
for sterilization
90% Ethylene oxide and 10% C02 require low pressure inside the area for
sterilization.
800 mg-1200mg/L concentration required for sterilization
Mechanism: alkylation of –OH, -SH, -COOH, -NH4 etc in proteins, nucleic acids and etc.
Disadvantages:- desorption time is more. Forced desorption has to be done with inert
sterile gases or sterile air.
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
1. A microscopic examination of culture isolate revealed spherical bodies with a
smooth outline growing in long chains, identify the organism.
A. Staphylococcus aureus
B. Streptococcus pyogenes
C. Rhyzopus stolonifer
D. Bacillus subtilis
2. Which of the following statements is correct for Gram positive bacteria
A) Cell wall has a thin peptidoglycan layer
B) Cell wall lipid content is very low·and smaller volume of periplasm
C) Lipopolysaccharide layer is present
D) Teichoic·acid is present
3. Which of the following method is useful for measuring the number of viable cells in culture?
A) Plate count technique
8) Dry weight method
C) Petroff-Hauser counter
D) Light scattering in a spectrophotometer
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RIPERAUTONOMOUS
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Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
4. Study the following four statements:[P] : Gram negative bacteria produce potent pyrogenic substances called endotoxins
[Q]: Ethylene oxide mixed with carbon dioxide or fluorinated hydrocarbons is used in gas
sterilization
[R]: D value is the time (for heat or chemical exposure) or the dose (for radiation exposure)
required for the microbial population to decline by one logarithmic unit
[S] : Spores of Geobacillus stearothermophilus {Bacillus stearothermophilus) are used for
sterility testing of moist heat sterilization process
Choose the correct answer.
(A) P, Q & R are correct but S is incorrect
(B) Q, R & S are correct but P is incorrect
(C) R, S & P are correct but Q is incorrect
(D) P, Q, R & S all are correct
5. By which mechanism the microorganisms are killed by autoclaving?
(A) Coagulation of the cellular proteins of the microorganisms
(B) Alkylation of essential cellular metabolites of microorganisms
(C) Stopping reproduction of microorganism cells as a result of lethal mutations
(D) Oxidation of RNA of microorganisms
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RIPERAUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
6. In relation to sterilization, what is the meaning of D 300F - 2 minutes?
(A) Death of all microorganisms in 2 minutes
(B) Death of 300 microorganism in 2 minutes
(C) Death of all microorganism in 2 minutes at 300°F
(D) Death of 90% microorganism in 2 minutes at 300°F
7. A given Gram-positive bacterium is differentiated from Gram-negative bacteria by
Gram staining. This is because its cell wall contains.
(A) Lysozyme
(B) Teichoic acid
(C) Membrane proteins
(D) Lipid A
8. Microorganism Typical characteristics
P. Corynebacterium diptheriae 1. Cells divide in three planes in an irregular pattern,
producing‘bunches’
Q. Streptococcus pyogenes 2. Cells are lined side by side like matchsticks and at
angles to one another
R. Staphylococcus aureus 3. long, branched, multinuclear filaments called ‘hyphae’
S. Streptomyces viridochroma 4. cells divide in one plane and remain attached to form
chain
(A) P-3,Q-4,R-2,S-1 (B) P-4,Q-1,R-2,S-3 (C) P-2,Q-4,R-1,S-3 (D) P-3,Q-2,R-1,S-4
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RIPERAUTONOMOUS
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Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
10. A technician is attempting to sterilize a plug of cotton in hermetically sealed condition in a glass
bottle by autoclaving, which of the following statements is correct?
A. It should be sterilized at 115-118 ℃for 30 minuetsB. It should be sterilized at 121 to 124 ℃ for 15 miutes at 15 lbs/sq. inch pressureC. Sterilization cannot be achieved.
D. It should be autoclaved at 126-129 ℃ with saturated steam for 10 Min..
11. Chemotaxis is a phenomenon that refers to
A. Directed movement in response to a chemical stimulus
B. Taxonomic classification of biochemicals
C. Large in flux of a chemical molecule within bacterial cells
D. Adherence of bacterial proteins to host cells.
9. Gram positive bacteria typically contain
A. Cell walls that lack peptidoglycans
B. Repeating units fo arabinogalactan and mycolates in their cell walls.
C. Peptidoglycan containing muramic acid and D-amino acids in their cell
walls
D. Cell walls containing predominantly polysaccharides and glycoproteins
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Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
12. For thermophillic microbes, the minimum growth temperature required is
A. 20℃B. 37℃C. 45℃D. 65℃
13 Obligatory anaerobes
A. Can tolerate oxygen and grow better in its presence
B. Do not tolerate oxygen and die in its presence
C. Can grow in oxygen levels below normal
D. Can grow in presence of atmospheric oxygen
14. Plasmid is a
A. Macromolecule involved in the protein synthesis
B. Circular piece of duplex DNA
C. A hybrid DNA that is formed by joining pieces DNA
D. Endogenous substance secreted by one type of cell
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Raghavendra Institute of Pharmaceutical Education and Research - AutonomousK.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Thank You