QP code:780401
I Examination
F.Y.B.Sc. Biotechnology Semester
Paper:BasicLifeSciences-||Microbia|Techniques
Model Answers
Do
1.
Y
Q. 1.
fifteen
Decimal Reduction Time: Time in minu
or time in tinuitt tor the thermal
2.
not
usuallY
sPorefo
dea
gO%
e'
a population to doub|e in size
Generation time: The time taken for
3.
4'Exponentialpn,,"'Thephaseinwhichmicroorganismsgrowanddivideatmaxima|
rate
5'Distortion:Anaberration/defectinmicroscopicimagewhichrendersasquare
6.
7.
object as an image with curved sides'
Acidic stain: Eosin/Acid fuschin/Nigrosine
acid/salts of Aluminium' iron'
Mordant: Gram's todine/ lodine solution/tannic
coPPer,zinc,chromium
8.
5"di,;
Detergent:
lauryl sulfate/Catylpyridinium.chloride/soap
9.Anti-microbialA|dehyde:Formaldehyde/glutaraIdehyde
10. ChromoPhore
11. Chemotrophs
12. Vitamins/ growth factor
plate/pour
Hemocytometer count/spread
13. Microscopic methods- Breed's count/
plate/ Electronic coulter/ turbidostatic methods
14. NumericalaPerture
15. Nutrient agar/
15. PhotograPhs
17. Incineration
18. lodoPhors
19. Death Phase
20. Abbe condenser
Q2: a) Exp|ain :
4 marks each
.,
Simp|e,,and
,,
simple
ff'$n:ilnstrument
(8 Marks)
Compound,, microscope and their app|ications
glass held usuallv in
consisting of onty one lens or magnifving
an adjustable frame
oSimp|emicroscopesdonotgiveashighmagnificationasacompoundmicroscope.
oApp|ication:tomakeanenlargedormagnifiedimageofminuteobjectsfor
observations/ studY'
Compound microscoPe:
.
.
sets of lenses (1. Objective 2. EYePiece)
nn optical instrument consisting of Two
Stage,
Coarse and fine adjustment knobs,
mounted in a holder/body tube'
Condenser and Mirror
APPropriate diagram
.
Compound microscope gives much greater magnification of specimen due to
product of magnification by objective (definite number of times) and further by the
.
Application: for viewing minute objects such as bacteria/ tissue sections etc. by
eyepiece
making a magnified image
Q 2:
b)
Discuss
" Different types of Biological stains" with two examples from
any three
classes ( 7 amrks)
Any three classes from
o
o
o
o
o
Nitro stains : Picric acid, Martius yellow, Aurantia
Azo stains: Methyl orange, methyl red, Brilliant yellow,Congo red, evan's blue, Sudan
stains, trypan blue, vital red etc.
Anthraguinine stains: alizarin, alizarin red S., purpurin
Thiazole stains:Thioflavin S, geranine G, primuline, yellow G etc.
o
Quinonimine stains (lndamines, indophenols, thiamines, oxanines, azines): neutral
red, neutral violet etc.
Phenyl methane stains (di/tri phenyl methane, diamino/triamino phenyl methane/
hydroxy phenyl methane, diphenyl-methyl methane): Brilliant green, Fast green,
o
Xanthene stains (pyronine, rhodamine, fluorane, phenolphthalein, sulfopthalein,
Methyl blue, methyl green Crystal violet, basic fuchsin, acid fuchsin etc.
B, Eosine, Erythrosine, fluorescein,
bromophenols, bromocresols, phenolphthalein, phenol red, acridine yellow/orange
acridine): PyronineB/Y, Rhodamine
o
etc.
Natural stains: Indigo, Indigo carmine, Cochineal, Carmine, Orcein, Orcinol,
LitmusBazilin, Hematoxylin etc.
Q 2: c) Describe the difference between " Light microscope" and "Dark field Microscope,,
( 8 marks) Minimum 4 correct four points
Light Microscope
o
o
The background of image in bright
Abbe condenser
o
Allows light
o
specimens
Used to view the objects visible in
to
pass through the
preparations)
o Contrast between the object and the background is
improved using a dark background /The specimens
appears bright on a dark background
o Dark-field stop in the condenser/ high aperture
darkfield condenser (Abbe/paraboloid/cardiod)
o Cone of light normally illuminating the object does
not enter the objective, only the light scattered or
object
a bright field
Dark field microscope
(fixed/stained
reflected by the specimen seen by the objective
to view the transparent/semitransparent
objects which are not readily visible in a bright field
o Used
principle o{Q 2: d) elaborate the
;l
lnm:,tmlu,:ru:i
differences in the
functions of
"f r1m staining' & State the
:1ffi:'ltive
an
d
physicochemicar properties
G ra
m
ne
gative orga
ni sm s
of the cell wall utt'
staining
is ba sed
!^Y.^
o
n th e
:::t::"'
peptidog|ycan.,o,,tinrcngnetworkandlipidcontent.Cel|wallsofGramnegativeorgan|sms
arethinner,have35%phospho|ipidsanaonty5-t5%peptidoglycanwithloosenetwork'
Hence,crystalviolet-iodinecomplexformedau..ingthestainingisretainedbyGrampositive
organismsandappearpurpleevenafterde-colourisationusingalcohol,whereasGram
negativeorganismsaredeco|ourised.Counterstainingwitheosin/saffraninmakesGram
negative organisms appear
pink'
(3 marks)
( marks)
Staining solutions: 4
Stains all the cells violet/purple
CrystalViolet; Basic stain:
vio|et. iodine (CVl)
tt,'" stain by forming crysta|
solution: Mordant ti"",
o
o
Gram,s iodine
comPlex
cells which
oAlcohol:deco|ouriser:Removesthestainse|ectivelyfromGramnegativece|ls
l".otou'i'eo ( Gram negative )
ii"
,,r,n,
counterstain,
o Eosin/saffranin:
purple colour'
positive organisms retain the
appear pink 'Gram
Q.3a)Describeanyh^,omethodsbasedonUseofheatfordestructionofmicroorganisms
( 8 marks)
( 4 Marks each)
Any two methods in detail:
pressure: Autoclave
Moist heat/Stem under
:Oven
Dry heat /hot air sterilization
o
.
o
e
o
lncineration
inspissator
Tyndalization/fractional sterilization:
Pasteurization
of an ideal ch-emical anti-microbial
Q. 3b) Discuss properties
properties from:
agent ( 7 marks)
any seven major
Expected answer to explain
Antimicrobial activitY
1'
2' SolubilitY
3. StabilitY
4. Non-toxicity to human and other animals
5. HomogeneitY
organic material
6. Non-combination with extraneous
temperature
7. Toxicity to microorganisms at room or body
8' CaPacitY to Penetrate
9. Non-corroding and non-staining
10. Deodorizing
11. Detergent caPacitY
12. AvailabilitY
Q.3c)Fi|trationcanbeusedse|ective|yforcontro|ofmicroorganismsforvarious
(8 marks)
appti"aiions ' lllustrate with examples
fi|ter, diatomaceous earth
materials -Asbestos pad in Seitz
different
of
fi|ters
Bacterio|ogical
and sintered glass disks in other
chamberland-pasteur filter
Berkefeld filter, porcelain in
in
filters.
Membrane or molecular filter - biologically inert cellulose esters
Applications:
Air sterilization : HEPA filter in laminar air flow
proof filters
sterilization of heat sensitive media and biological fluids - Bacteria
Water filters etc.
q. 3d)
Explain applications
of heavy metals and dyes as anti-microbial agents with
examples (7 Marks)
microorganisms ( 4 marks)
Heavy metals and their compounds have detrimental effects on
Examples: (anY 2 examPles)
Mercurv:
Ammoniated mercury- Used in
Inorganic compounds : Mercuric chloride, Mercuric oxide,
ointments disadv: toxicity and corrosive action
less irritant and less
organic compounds: mercurochrome, metaphen, merthiolate,mecresin:
surfaces
toxic; used as antiseptic preparations for cutaneous and mucosal
Silver
:
picrate- Used as antiseptic colloidal silver compounds: silver nitrate, silver lactate, silver
silver released from the combination
Bacteriostatic/bacteriocidal effects depend on the free
a gonococcal infection of eyes'
Silver nitrate widely used to prevent opthalmia neonatorum,
Copper:
.-_^r :_ ^...i_. .
than bacteria-used in swimming
molds
and
algae
against
effective
More
sulfatecopper
to prevent certain plant
pools and open water reservoirs; Bordeaux mixture - afungicide
diseases
Dyes anY two examples
(3 marks)
the culture media on the basis of composition and
Q.4A) Define culture medium. classify
state any one use of each (8 marks)
preparation used to grow, transport or store
Ans: The culture medium is a solid or liquid
micro organisms.
Classification of medium on the basis of composition:
which all the components are known is called
1) Synthetic or Defined medium: Medium in
Synthetic or Defined medium'
It is use to Chemotrophs and heterotrophs'
of unknown chemical
2) Complex medium: Medium which contains some ingredients
a simple complex medium may be sufficiently rich
composition. such media are useful as
of many different micro organisms'
and complete to meet the nutritional requirements
To grow fastidious organisms'
the organisms'
S) Selective medium: Medium is used for selecting
as it contains Na taurocholate'
bacteria
enteric
growth
of
Mac conkey's agar selects the
from a group of organisms'
organism
single
4) Differential medium: Medium differentiates
organisms'
hemolytic
non
and
Blood agar is used to differentiate between hemolytic
q'4
B) How would you cultivate microorganisms by chemostat
and tubidostat (7 marks|
chemostat: a chemostat contains sterile medium is fed into the
culture vessel at the same
rate as the media containing micro organisms is removed. The
culture medium for the
chemostat possesses an essential nutrients in the limiting
quantities. Because of the
presence of limiting nutrients, the growth rate
is determined by the rate at which new
medium is fed into the chamber and the final cell density depends
on the concentration of
the limiting nutrient.
The rate of nutrient exchange is expressed as the dilution
rate. lf teg dilution rate rises too
high the organisms can actually be washed out of the
culture vessel before reproducing
because the dilution rate is greater. At very low dilution rate
an increase in cell density and
growth rate occurs. (4 marks)
Turbidostat: lt has a photocell that measures the absorbance or turbidity
of the culture in
growth vessel. The flow rate of media through the vessel
is automatically regulated to
maintain a pre determined turbidity or cell density. lt provides a continuous
supply of
organisms in exponential phase. lt make possible to study the growth
of organism,'und",.
limiting nutrient supply. (3 marks|
q.4.c) write a detailed account on measurement
of growth of microbes ( 7 marks)
Ans: 1) Measurement of cell number:
It can be done by Breed's count in which lsq. cm area sguare is marked on
Grease free slide.
Thin smear is made on slide. The smear is stained with crystal violet for 1min.
Drain off the
stain. Rinse the slide with d/w. observe and count organisms under oil immersion
lens.
Cell suspension is loaded on Hemocytometer slide. Either organisms in WBC
chamber
under
low power or RBC chamber under high power are counted.
2) Measurement of cell mass: The cell mass can be measured by taking the packed cell
volume of cells. The wet weight and dry weight of the cells is calculated. Spectrophotometer
and colorimeter can be used to enumerate micro-organisms on the basis of turbidity of
culture.
Q.4 D) How would you isolate pure culture by spread plate and streak plate techniques (7
Marks)
Ans: Can be explained by drawing the techniques
as
well
lsolation of pure culture by spread plate Method:
1. Suspension containing mixture of organisms is serially diluted.
2. Desired dilution amongst are selected.
3. 0.1 ml of the culture is added to sterile nutrient agar plate and spread on the surface of
agar till the surface is dry, under sterile conditions.
4. Plates are incubated at desired temperature for desired time period depending on the
nature and type of organisms.
5. After incubation time, well distributed and separated colonies appear.
lsolation using streak plate:
1. The method involves-
T- streak method: In this method, the sterile nutrient agar plate is divided into 3 zonesinoculation, sterile and isolation zone. A loopful of the culture sample is inoculated in
inoculation zone and the culture is brought down perpendicularly in the isolation
zone,where it is spread with the sterile nichrome Loop. Observe for the appearance of
isolated colonies in the isolation zone after the end of incubation period.
The other method is the Hexagonal method. In this method the medium plate is divided into
6 qudrants, taking care that the first and the sixth quadrants are not joint. lsolated colonies
appear in the centre ofthe plate.
Q.5 Short notes {any three)
a) Halogens for microbial population control:
lodine : Oldest and most effective germicidal agent
ncture iodine-several preparations
lodophors-polyvinyl pyrrolidone (pvp-l) - non-staini ng and low irritant properties
Applications: anti-bacterial, sporicidat, fungicidal, virucidal disinfectant;
for skin; water; air;
food utensils etc.
Chlorine and chlorine compounds
Chlorine gas: purification
Hypochlorites: calcium hypochlorite, sodium hypochlorites
Chloramines: monochloramine, chloramines-T, azochloramide
Sanitizing agents, antiseptics
Ti
b) FDA method for evaluation of water soluble chemical disinfectant:
Phenol-coefficient method is for determination of effectiveness of the test disinfectant
(water soluble chemical disinfectant) as compared with that of pure phenol under
the
carefully standardized conditions. lt is based on cornparison of anti-microbial effects of the
test disinfectant and phenol in liquid media inoculated with the same test microorganism
under the same conditions.
General procedure:
o
o
o
o
o
o
o
o
o
Series of dilutions of the test disinfectant
Similar series of dilutions of pure phenol of 1:90,1:90 and 1:100
Temperature 2OoC
Inoculate 0.5 ml of standard young broth culture of the test microorganism
At regular time intervals, a loopful is transferred to 10 ml sterile broth
After 48 hours incubation at370c, growth in the broth tubes is recorded
lf all the organisms are killed : No growth in the corresponding subculture
Test is repeated with higher dilution of the disinfectant such that not all the
organisms are killed in some of the tubes
Phenol coefficient is calculated as the ratio of the highest dilution of the test
disinfectant not killing the organisms in five minutes (indicated by growth in the
corresponding subculture) but killing in ten minutes (say it is 1:450) to the
corresponding dilution of phenol (say it is 1:90, then Phenol coefficient for that
disinfectant would be 450/90=5).
c) Zeil-Neelsen staining/ Acid fast staining:
Differential staining used especially for staining Tuberculosis bacilli (Mycobacterium
tuberculosis) and related organisms (genus: Mycobacterium) which have acid fast waxy
material (mycolic acids) in the cell. The method is based on the relative solubility of the
stains. Fuchsin is more soluble in phenol than in water or acid alcohol. phenol is more
soluble in lipids/wax (that is present in the tubercule bacilli) than in water. Phenol with red
fuchsin enters the cell lipids and remains even after treatment with decolourizing acidalcohol.
Thin smear of cells /sample (i.e. sputum) is dried, heat-fixed and covered by carbol fuchsin
solution and heated to gOoC over a steam bath for 4 minutes. This softens wax and the oye
penetrates. Excess dye is washed off and smear is treated with alcohol containing s-i:O%
hydrochloric acid to decolorize every other objects than the acid-fast bacteria. Methylene
blue or Brilliant green is then applied as a counter-stain. Acid fast organisms appear bright
red against the blue/green field.
d) Arithmetic growth
In certain abnormal condition s, the kinetics of bacterial growth may become arithmetic
rather than exponential. A number of conditions can lead to arithmetic growth. For
eg. lf a
bacterium requires nicotinic acid as a growth factor is deprived of this
nutrient, it is unable
to synthesize pyridine nucleotide of which nicotinic acid is a specific biosynthetic precursor.
As a result of the functions of pyridine nucleotide in electron transport chain, their levels
in
cell determined the overall rate of metabolism and hence growth. When no further
synthesis of compound can occur, the growth rate of the population becomes directly
proportional to the supply of pyridine nucleotide precursor in the cell. The rate of increase
of cells remains constant.
e) Phase -contrast microscope
o
Microscopy controls
'
o
cells/cytological details become visible
Use of annular diaphram in the focal lane of the substage condenser, which controls
illumination of the objects and phase shifting element/ phase plate
The light reflected by the object is passed through phase altering plate resulting in
interference to reduce the illuminating rays in a systematic manner
.
o
the contrast in the image in such a way that
unstained
Appropriate ray diagram
Used to view unstained cells/cytological details , advantage of accurate/ real-time
viewing of unstained living cells /movements/propagation/exchange of cellular
constituents and other processes etc.