i

si
Teim, AY :oi: - :oi
Plai i Lab ] Plaimaceuiical Miciobiology
Micioscopy 8 Miciomeiiy ] i
Cliisiine }o Ann M. Zuiga, BS IP

Exeicise No. i
MICROSCOPY AND MICROMETRY

!"#$%&"%'%() - siudy of !"#$%%$&'(")!)+
oiganisms noi seen by ile naled eye
bacieiia (ala animalcules), fungi, pioiozoans, lelminiles (paiasiiology), (viius - noi an oiganism)

*(+%( -'( .//01/(2%/3
- ,"-('. '.-, /"#$%,#%0.
- failei of miciobiology
- i
si
io desciibe animal cells

1%/0%2-3 /"#$%,#%0. - an opiical insiiumeni used io obseive ilings ilai aie beyond oidinaiy vision
1%/0%2-3 '"(45 /"#$%,#%0. - piecision micioscope ilai uses iwo seis of lenses, !"#$%& and !'()"*+,), and employs
ligli as iis souice of illuminaiion

Magnificaiion is aclieved as follows:
illuminaioi ! condensei ! specimen ! objeciive lens ! oculai lens |+-,)&*). image is foimedj

456/) %7 8"#$%)#%6/

6789:; <8=>? ?@7A <8=>? <>BC7=D1=E;
F:@D= G
1CE;7@D;
=>=1;7CE
0)1"&+2*+!-
Biigli
baclgiound
Has a filiei
Fluoiescence dye;
absoibs eneigy of
sloii ligli waves
(blue ligli); dye
emiis a ligli of a
longei " (gieen
ligli)
Densei paiis -
appeai biigli

Wiil densiiy
close io H
:
O -
appeai dail

3%4-+5+"%*+!- iooo - :ooo iooo - :ooo iooo - :ooo iooo - :ooo :ooK - (ooK
Useful foi
unsiained
specimen
Peimiis gieaiei
coniiasi
;=!
H;$I-,/",,"%-
='.#5$%-
!"#$%,#%0)J
! slice micio -
oiganisms
inio seciions
! impoiiani
wlen viewing
inieinal
oigans
6.,%-*%4)1
Diieci ligli
souice is used
Foi viewing
12+&!"7)*)1
(spiial-slaped,
STD)
Rapid iesis ilai
ideniify disease-
causing
miciooiganisms
No need io use
siains
D=! HD#I--"-(
='.#5$%-
!"#$%,#%0)J
! piovides D
view
! impoiiani
wlen viewing
fungi
0+1%.,%-*%4)1
Siaining is
employed (may
aliei cell's
slape]size oi lill
ile cell)
Low illuminaiion Expensive Expensive

i
si
Teim, AY :oi: - :oi
Plai i Lab ] Plaimaceuiical Miciobiology
Micioscopy 8 Miciomeiiy ] :
Cliisiine }o Ann M. Zuiga, BS IP

8K !"#$%,#%0)
@K 1I$. %L 54. !"#$%,#%0.
i. Clean, diy 8 fiee fiom dusi; coveied wlen noi in use
:. Caiiying - aim 8 base
. Avoid jaiiing
(. Lenses: "fingeis, #lens papei
. "objeciives $ liquid, excepi ile immeision oil foi ile i.S-mm objeciive
6. Clean OIO immediaiely w] lens papei. Diied oil on ile objeciive - 89$!$ ilen lens papei!
y. Nevei foice adjusimenis.
S. Avoid unnecessaiy iuining of ile coaise 8 fine focusing lnobs.
q. "alcolol ] xylol $ lacqueied pioducis
io. Befoie ieiuining, see io ii ilai ile
a. LPO is in place
b. body iube is fully loweied
c. concave miiioi is iuined upside down

6K FI$5, %L 54. !"#$%,#%0.
i. 6I,. - ile boiiom poiiion on wlicl ile micioscope iesis
:. F"M%5 0%"-5 - enables one io iili aim baclwaids io adjusi ile eyepiece leigli
. D2&,5I(. '"(45 %$ /"$$%$ - ile souice of illuminaiion locaied ai ile base
o 79'+ - conceniiaied ligli ] aiiificial ligli
o #%(#'-/ - dispeised ligli ] naiuial ligli
,2&,5I(. "''2/"-I5%$ - las a voliage coniiol io vaiy ile iniensiiy of ligli
(. D5I(. - ile plaifoim on wlicl ile slide iesis wlile being viewed
- )+'&/ %6/("(&: allows ligli io pass fiom below iliougl ile objeci being examined
- io lold ile slide in posiiion foi viewing,
a) siage equipped wiil )6$"(& :)+'&/; #9"6)
b) #9'!6<+56/ !/#2'("#'9 )+'&/ - peimiis piecise movemeni of ile specimen
. 1%-3.-,.$ - a lens locaied beneail ile siage ilai conceniiaies ile ligli beam on ile specimen
6. ?"I04$I(/ '.M.$ - an aim aiiacled io ile condensei ilai iegulaies ile amouni of ligli passing iliougl
ile condensei
y. 1%I$,. I3N2,5/.-5 O-%& - a usually laigei lnob ilai iaises 8 loweis ile body iube (oi siage) io biing
ile specimen inio view
S. <"-. I3N2,5/.-5 O-%& - a usually smallei lnob found below oi exieinal io ile CAK; used foi fine oi
focal focusing
q. :.I3 %$ &%3) 52&. -ieceives ile oculai lens; suppoiis ile objeciive lens sysiem, wlicl is mounied on a
movable nosepiece
io. E%,.0".#. - a plaie, usually ciiculai ai ile boiiom of ile body iube; caiiies iliee objeciive lenses and
peimiis sequeniial posiiioning of ilese lenses ovei ile ligli beam passing iliougl ile siage opening
ii. D#I--"-( %&N.#5"M. HPQJ - sloiiesi objeciive; noi pieseni on all micioscopes
i:. >%R 0%R.$ %&N.#5"M. HSTQJ - also called ile => !! %?@/#+"-/; nexi sloiiesi objeciive
i. :"(4 0%R.$ %&N.#5"M. HUVQ %$ UPQJ - also called ile A !! %?@/#+"-/ 8 2"&2<B$5 %?@/#+"-/; :
nd
longesi
objeciive
i(. C"' "//.$,"%- %&N.#5"M. HSTTQJ - also called ile =CD !! %?@/#+"-/; disiinguisled by an eicled coloied
ciicle; longesi objeciive
! 7"E/B %"9 oi #/B'$ 1%%B %"9 is used
! As ile magnificaiion incieases, ile size of ile lens ai ile iip of ile objeciive becomes piogiessively
1:%$$)& and admiis $)11 ligli.
! Wlen ile oil immeision lens is used, immeision oil fills ile space beiween ile objeciive and ile
specimen. Because immeision oil las ile same iefiaciive index as glass, ile loss of ligli is
minimized.
i
si
Teim, AY :oi: - :oi
Plai i Lab ] Plaimaceuiical Miciobiology
Micioscopy 8 Miciomeiiy ]
Cliisiine }o Ann M. Zuiga, BS IP

i. C#2'I$ W =).0".#. HSTQJ - a iemovable lens ai ile iop of ile body iube; someiimes fiiied wiil a poiniei
oi measuiing scale

1K <%#2,"-( I D0.#"/.- - expeiimeni piopei

88K !"#$%/.5$)
;%$+'&%*+!- !5 *7) 3+"&!1"!2)

C#2'I$ /"#$%/.5.$ - used io measuie micioscopic objecis
- a glass disc wiil a mounied scale
- inseiied inio ile eyepiece and musi be calibiaied foi ile paiiiculai objeciive, eyepiece and iube
lengil employed
D5I(. /"#$%/.5.$ - a glass slide wiil giaduaiions of lnown inieivals
- ile lengil of one small division is o.oi mm oi io pm (one big division is o.i mm oi ioo pm)

! i pm = i]iooo mm - i]:,(oo incl

A. 1I'"&$I5"%- %L 54. C#2'I$ !"#$%/.5.$
i. Sepaiaie ile eyepiece fiom ile diaw iube.
:. Unsciew lens covei of eyepiece.
. Inseii oculai miciomeiei.
(. Replace lens covei and ieiuin eyepiece io ile diaw iube.
. Lool inio ile eyepiece io see io ii ilai ile disc is noi inveiied.
6. Place siage miciomeiei on ile siage and focus on ile scale. Aiiange ile siage miciomeiei so ilai one
line on ii coincides wiil a line on ile oculai scale.
y. Lool acioss ile field foi anoilei line on ile oculai miciomeiei coinciding wiil a line on ile siage scale.
S. Couni ile numbei of divisions on ile oculai miciomeiei subiended by ile numbei of divisions on ile
siage miciomeie .
q. Compuie foi ile calibiaiion facioi using ile foimula:
!
calibration factor =
stage micrometer divisions subtended
ocular micrometer divisions subtended
value of one stage micrometer division

! CF
LPO
= CF
HPO

B. !.I,2$./.-5 %L 54. D0.#"/.-
Measuie ile specimens given io you by couniing ile numbei of division ii coveis on oculai
miciomeiei. Knowing ile "%$+'&%*+!- 5%"*!&, ile size of ile specimen may be compuied in m.




i
si
Teim, AY :oi: - :oi
Plai i Lab ] Plaimaceuiical Miciobiology
Micioscopy 8 Miciomeiiy ] (
Cliisiine }o Ann M. Zuiga, BS IP


lll. !"#$%&'($) (CallbraLlon of mlcroscope)

*#+,-$ &"#$%&'('$- use Lo measure mlcroscoplc ob[ecLs, a glass dlsc wlLh mounLed scale musL be callbraLed for
arLlcular ob[ecLlve
Lyeplece
1ube lengLh
! unlL of llnear measuremenL ls Lhe mlcromeLer (m)
! 1m =
1
1000 mm
=
1
25,400 nch


/(-0' &"#$%&'('$- a glass sllde wlLh graduaLlons of known lnLervals
! LengLh of one 1&-,, 2"3"1"%4 ls 5657&& 8 75&
! LengLh of one 9"0 2"3"1"%4 ls 567&& %$ 755&

:6 ;%< (% #-,"9$-(' -4 %#+,-$ &"#$%&'('$
1. SeparaLe eyeplece from draw Lube
2. unscrew lens cover of eyeplece
3. lnserL ocular mlcromeLer
4. 8eplace lens cover and reLurn eyeplece Lo Lhe draw Lube
3. Look lnLo Lhe eyeplece Lo see Lo lL LhaL Lhe dlsc ls noL lnverLed
6. lace sLage mlcromeLer on Lhe sLage and focus on Lhe scale
7. Arrange Lhe sLage mlcromeLer so LhaL one llne on lL colncldes wlLh a llne on Lhe ocular lens
8. Look across Lhe fleld for anoLher llne on Lhe ocular mlcromeLer colncldlng wlLh a llne on Lhe sLage scale
9. CounL Lhe number of dlvlslons on Lhe ocular mlcromeLer subLended by Lhe number of dlvlslons on Lhe sLage mlcromeLer
10. CompuLe for Lhe callbraLlon facLor uslng Lhe formula:

=-,"9$-("%4 >-#(%$ =
Stugc mcomctc dsons subtcndcd b ocuIu mcomctc vuIuc o] 1 stugc mcomctc dson
0cuIu mcomctc dsons subtcndcd b stugc mcomctc

8. MeasuremenL of Speclmen
1. Measure Lhe speclmens glven Lo you by counLlng Lhe number of dlvlslon lL covers on ocular mlcromeLer
2. knowlng Lhe callbraLlon facLor, Lhe slze of Lhe speclmen may be compuLed ln m

!"#$% '"()*+(*,-. /01 2$3 "',*)%04% ,0)%+ *5 0 (*''*4 6"#$%
'"()*+(*,3. /71 8*9 "''3)+"*4 *"6 #0%$3)+ '*)3 6"#$% 5*) :+3 "4
%$3 '"()*+(*,3.
!"#$%
HarleyPrescott:
Laboratory Exercises in
Microbiology, Fifth Edition
I. Microscopic Techniques 1. BrightField Light
Microscope and
Microscopic Measurement
of Organisms
The McGrawHill
Companies, 2002
fication or the focal length. The latter is about equal
to or greater than the working distance between the
specimen when in focus and the tip of the objective
lens. For example, the low-power objective is also
called the 10, or 16 millimeter (mm), objective; the
high-dry is called the 40, or 4 mm, objective; and
the oil immersion is called the 90, 100, or 1.8 mm
objective. As the magnification increases, the size of
the lens at the tip of the objective becomes progres-
sively smaller and admits less light. This is one of the
reasons that changes in position of the substage con-
denser and iris diaphragm are required when using
different objectives if the specimens viewed are to be
seen distinctly. The condenser focuses the light on a
small area above the stage, and the iris diaphragm con-
trols the amount of light that enters the condenser.
When the oil immersion lens is used, immersion oil
fills the space between the objective and the specimen.
Because immersion oil has the same refractive index
as glass, the loss of light is minimized (figure 1.1). The
eyepiece, or ocular, at the top of the tube magnifies
the image formed by the objective lens. As a result, the
total magnification seen by the observer is obtained by
multiplying the magnification of the objective lens by
the magnification of the ocular, or eyepiece. For exam-
ple, when using the 10 ocular and the 43 objective,
total magnification is 10 43 = 430 times.
Procedure for Basic Microscopy: Proper Use
of the Microscope
1. Always carry the microscope with two hands. Place
it on the desk with the open part away from you.
2. Clean all of the microscopes lenses only with
lens paper and lens cleaner if necessary. Do not
use paper towels or Kimwipes; they can scratch
the lenses. Do not remove the oculars or any other
parts from the body of the microscope.
3. Cut a lowercase e from a newspaper or other
printed page. Prepare a wet-mount as illustrated in
figure 1.2. Place the glass slide on the stage of the
microscope and secure it firmly using stage clips.
If your microscope has a mechanical stage device,
place the slide securely in it. Move the slide until
the letter e is over the opening in the stage.
4. With the low-power objective in position, lower
the tube until the tip of the objective is within
5 mm of the slide. Be sure that you lower the tube
while looking at the microscope from the side.
5. Look into the microscope and slowly raise the
tube by turning the coarse adjustment knob
counterclockwise until the object comes into
view. Once the object is in view, use the fine
adjustment knob to focus the desired image.
6. Open and close the diaphragm, and lower and raise
the condenser, noting what effect these actions
have on the appearance of the object being viewed.
Usually the microscope is used with the substage
condenser in its topmost position. The diaphragm
should be open and then closed down until just a
slight increase in contrast is observed (table 1.1).
7. Use the oil immersion lens to examine the stained
bacteria that are provided (figure 1.3ad). The
directions for using this lens are as follows: First locate
Bright-Field Light Microscope and Microscopic Measurement of Organisms 3
Figure 1.1 The Oil Immersion Objective. An oil immersion
objective lens operating in air and with immersion oil. Light rays
that must pass through air are bent (refracted), and many do not
enter the objective lens. The immersion oil prevents the loss of
light rays.
Figure 1.2 Preparation of a Wet-mount Slide. (a) Add a
drop of water to a slide. (b) Place the specimen (letter e) in the
water. (c) Place the edge of a coverslip on the slide so that it
touches the edge of the water. (d) Slowly lower the coverslip to
prevent forming and trapping air bubbles.
(a)
(c) (d)
(b)
Air Oil Cover
glass
Slide
HarleyPrescott:
Laboratory Exercises in
Microbiology, Fifth Edition
I. Microscopic Techniques 1. BrightField Light
Microscope and
Microscopic Measurement
of Organisms
The McGrawHill
Companies, 2002
This numerical value holds only for the
specific objective-ocular lens combination used
and may vary with different microscopes.
6 Microscopic Techniques
Stage
micrometer
Superposition of scales allows
calibration of ocular scales
(10 ocular units = 0.07 mm)
(d)
(c)
Ocular
micrometer
Image of ocular micrometer
with uniformly spaced lines
Image of stage micrometer
with uniform lines at standard
known intervals
Space =
0.01 mm
0.1 mm
0 100 20 40 60 80
0
2
0
4
0
6
0
8
0
0 100 20 40 60 80
(a) (b)
Figure 1.4 Calibrating an Ocular Micrometer.
HINTS AND PRECAUTIONS
(1) Forcing the fine or coarse adjustment knobs on the mi-
croscope beyond their gentle stopping points can render
the microscope useless. (2) A general rule for you to note
is that the lower the magnification, the less light should be
directed upon the object. (3) The fine adjustment knob on
the microscope should be centered prior to use to allow
for maximum adjustment in either direction. (4) If a slide
is inadvertently placed upside down on the microscope
stage, you will have no difficulty focusing the object
under low and high power. However, when progressing to
oil immersion, you will find it impossible to bring the ob-
ject into focus. (5) Slides should always be placed on and
removed from the stage when the low-power (4 or 10)
objective is in place. Removing a slide when the higher
objectives are in position may scratch the lenses. (6) A
note about wearing eyeglasses. A microscope can be fo-
cused; therefore, it is capable of correcting for near- or
farsightedness. Individuals who wear eyeglasses that cor-
rect for near- or farsightedness do not have to wear their
glasses. The microscope cannot correct for astigmatism;
thus, these individuals must wear their glasses. If eye-
glasses are worn, they should not touch the oculars for
proper viewing. If you touch the oculars with your
glasses, they may scratch either the glasses or the oculars.
(7) Because lens cleaner can be harmful to objectives, be
sure not to use too much cleaner or leave it on too long.
The distance between the lines of an ocular microme-
ter is an arbitrary measurement that has meaning only if
the ocular micrometer is calibrated for the specific objec-
tive being used. If it is necessary to insert an ocular mi-
crometer in your eyepiece (ocular), ask your instructor
whether it is to be inserted below the bottom lens or
placed between the two lenses. Make sure that the etched
graduations are on the upper surface of the glass disk that
you are inserting. With stained preparations such as
Gram-stained bacteria, the bacteria may measure smaller
than they normally are if only the stained portion of the
cell is the cytoplasm (gram-negative bacteria), whereas
those whose walls are stained (gram-positive bacteria)
will measure closer to their actual size.
Calibrate for each of the objectives on your
microscope and record below. Show all
calculations in the space following the table; also
show your calculations to your instructor.
Low power (10 objective) 1 ocular space = ______ mm
High-dry power (40 objective) 1 ocular space = ______ mm
Oil immersion (90 objective) 1 ocular space = ______ mm