andhra pradesh state govt. apsche jntua jntuk · 2020. 7. 15. · gpat online classes...

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)

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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR



Sheaths- cells enclosed in hollow tube

Prosthecae/stalks- semi rigid extension of cell wall and cytoplasmic membrane.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR



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)

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR



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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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)

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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℃

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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.

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


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

RIPERAUTONOMOUS

NAAC &

NBA (UG)

SIRO- DSIR


Thank You

andhra pradesh state govt. apsche jntua jntuk · 2020. 7. 15. · gpat online classes...

Documents