T HE C O MP O U N D MIC R O S C O P E

 compound microscope is an optical tool that is used to observe things

that are beyond ordinary vision
 is one of the basic instruments of a microbiologist
 the term compound microscope is derived from the fact that the specimen
is magnified twice, first by the objective and second by the eyepiece
 designed to magnify objects as desired but the resolution is limited by the
wavelength of light used to illuminate the specimen
PARTS AND FUNCTIONS
BASE
 keeps the microscope steady at any position of the stage
ARM
 fastened to the base through the inclination joint
 permits the adjustment of the stage to the desired angle
CONCAVE MIRROR
 it reflects the light into the condenser
IRIS DIAPHRAGM
 regulates the amount of light entering the condenser
 below the diaphragm is a slot to accommodate different types of light
filters IMPORTANT TERMS IN MICROSCOPY
CONDENSER MAGNIFICATION
 concentrates the light rays received from the mirror and sends them to the  how much larger the object appears compared to its actual size
objective RESOLVING POWER
STAGE  a measure of the clearness of the appearance of the specimen
 is the horizontal platform upon which the specimen to be examined in  it is the minimum distance two points can be separated and still be
placed distinguished as two separate points. For instance, what appears as one
 At the center of the stage is a circular aperture star in the sky can be resolved as twin stars with the use of a telescope
SPRING CLIPS  resolving power of the human eye is limited just as the resolving power of
 hold the slide in place on the stage the telescopes and microscopes is limited as well.
OBJECTIVE TYPES OF MICROSCOPES
 part of the optical system of the microscope which produces the COMPOUND
specimens initial magnified image (real) within the body tube
 are light illuminated
 student microscope has three objectives: a dry low power, a dry medium
 image seen with this type of microscope is two dimensional
power, and an oil immersion high power objective
 the most commonly used
REVOLVING NOSEPIECE
 can view individual cells, even living ones
 where the three parfocal objectives are attached
 has high magnification but has a low resolution
 allows convenient exchange of the objectives
BODY TUBE  the source of radiation for image formation is visible light
 body tube is a hollow cylindrical tube through which the light passes from  uses air as a medium
the objectives to the eyepiece  uses glass slides as special mounting
DRAW TUBE  the nature of lenses is glass
 the upper position of the body  it uses mechanical focusing
EYEPIECE OR OCULAR  it is enhanced through changing the objectives
 part of the optical system through which the specimen is viewed  provides the specimen’s contrast through light absorption
 intermediate image projected by the objective is enlarged by the eyepiece DISSECTION OR STEREOSCOPE
VIRTUAL IMAGE  are light illuminated
 final image formed  image seen with this type of microscope is three dimensional
MAGNIFICATION  used for dissection to get a better look at larger specimen
 the product of the magnifying power of the objective and the eyepiece  cannot view individual cells, even living ones
 if the magnifying power of the objective is 100x and the eyepiece is 10x,  has low magnification
the total magnification will be 1000x  the source of radiation for image formation is visible light
FINE FOCUS KNOB  uses air as a medium
 used for maximum definition  uses glass slides as special mounting
 slow but precise control used to fine focus the image when viewing at the  the nature of lenses is glass
higher magnifications  it uses mechanical focusing
COARSE FOCUS KNOB  usually have one objective
 used to bring the object into focus  provides the specimen’s contrast through light scattering or light reflection
 a rapid control which allows for quick focusing by moving the objective CONFOCAL
lens or stage up and down  uses laser light
 is used for initial focusing  this light is used because of the wavelength
 scan across the specimen with the aid of scanning mirrors
 image is then placed on a digital computer screen for analyzing
 the source of radiation for image formation is laser light
 uses air as a medium
 uses glass slides with dyed samples as special mounting
 the nature of lenses is glass lenses with dichromatic mirrors
 it uses digital computer motorized focusing mechanism
 it is digitally enhanced
 provides the specimen’s contrast through laser light with dichromatic
mirror concentrated at pinhole
SCANNING ELECTRON
 uses electron illumination
 image seen with this type of microscope is three dimensional
 has high magnification and high resolution
 specimen is coated in gold and the electrons bounce off to give you an
exterior view of the specimen
 pictures are in black and white
 the source of radiation for image formation is electrons
 uses vacuum as a medium
  uses aluminum stubs and are coated in gold as special mounting Bacteria 0.5 – Unicellular Absent Asexual Flagella
 the nature of lenses is one electrostatic and some few electromagnetic 10 µm
lenses Yeast 5- Unicellular Present Asexual Non-motile
 it uses electrical focusing and adjusting 100µ
 provides the specimen’s contrast through electron scattering m
TRANSMISSION ELECTRON Molds 10µm Multicellular Present Asexual Non-motile
 uses electron illumination –
 image seen with this type of microscope is two dimensional 10cm
 has high magnification and high resolution Algae 0.2 to Both Present Both Flagella
 specimen is sliced into thin slices and the electrons beams pass through 2µm
this up to
 the source of radiation for image formation is electrons 60m
 uses vacuum as a medium Protozoa 1 to Unicellular Present Asexual Cell
 uses thin films of collodion or other supporting materials on copper grids 150 extension,
as special mounting µm flagella,
pseudopo
 the nature of lenses is one electrostatic and some few electromagnetic
dia
lenses
 it uses electrical focusing (current of the objective lens coil) and adjusting
(changing current of the projector lens coil)
 provides the specimen’s contrast through electron scattering
THREE FORMS OF BACTERIA
CHARACTERISTICS OF THE MICROSCOPE COCCUS
Features Low Medium High Power  spherical
Power Power (dry) (immersion oil)  usually found in clusters or chains
(dry)  does not dry out quickly
Focal length (mm) 16mm 4mm 1.8-2.0mm  absorb nutrients slowly
Working distance 4-8mm 0.5-0.7mm 0.1mm BACILLUS
(mm)  rod-shaped bacteria
Linear Magnification 10x 40-45x 90-100x  has large surface area which helps them to take in nutrients faster
(X) SPRILLUM
Numerical aperture 0.25-0.30 0.55-0.65 1.25-1.4  a spiral bacterium
(N.A.)  has flagella at both ends which allow them to move like a corkscrew and
Diameter of front 2.0mm 0.4mm 0.2mm made them capable of moving faster than the other bacteria
lens (mm) THREE FORMS OF LOCOMOTIVE ORGANS
CILIA
INVERSION OF IMAGES
 the largest group
 are hair-like projections
 protrude from the pellicle and the membrane that surrounds the organism
 used to beat against the fluid environment
FLAGELLA
 move themselves by whipping their flagellum back and forth or in a
corkscrew motion
PSEUDO/AXO/PODIA
 involves the flow of cytoplasm as extensions of organism
 responsible for amoeboid movement, a sliding or crawlinglike form of
Letter “e” as seen by the unaided eye locomotion
A SE P T IC T E C H N I QU E
 pure culture technique has resulted in the isolation and classification of
organisms responsible for infections, fermentations, nitrogen fixation, and
the like
 further advances in the different areas in microbiology will depend upon
studies of individual microorganisms in pure cultures
 since most of the microbiological studies utilize pure cultures, the culture
media should be sterilized and maintained in sterile condition
 this sterile medium when inoculated with a pure culture of microorganisms
Letter “e” as seen under the microscope should be free from outside contamination
 procedures which prevent the entrance of unwanted microorganisms are
MOVEMENT OF THE SPECIMEN ON STAGE AND ITS commonly referred to as Aseptic Techniques
CORRESPONDING MOVEMENT IN THE EYEPIECE
If the letter e on stage Is moved to The image in the eyepiece is moved
to: ASEPTIC TECHNIQUE PROCEDURE
3 o’clock 9 o’clock INOCULATION
6 o’clock 12 o’clock  to grow in a sterile medium, several cells (the inoculum) are transferred
9 o’clock 3 o’clock (inoculated) into the medium using special precautions to maintain the
12 o’clock 6 o’clock purity of the culture being transferred
STERILIZATION OF THE WIRE LOOP
O B S E R V IN G MI C R O OR GA N I SM S a. hold the wire loop like a pencil and heat both the loop and lower part of
the handle over the flame of an alcohol lamp or burner. For faster heating,
 they exhibit diverse characteristics morphologically and nutritionally
hold the loop almost vertically
 variations can be found in their shape, cellular structure, types of motility, b.allow the loop to cool a bit – do not let it in contact with anything
and size TRANSFERRING OF CULTURE FROM ONE TUBE TO ANOTHER
 can be autotrophic and heterotrophic; chlorophyll-free and chlorophyll a.hold the tube with the left hand
containing; unicellular and multicellular; saprophytic and parasitic; and b.sterilize wire loop
ranging from bacteria to algae, fungi and protozoa c. remove the plugs of the tubes as follows:
 some concepts of diversity of morphological types among organisms can be i. grasp the plug of the culture tube with the little finger of the right hand
gained by careful microscopic examination of such specimens ii. hold the plug of the second tube between the small and ring fingers
Micro- Relative Uni or Nucleus Reproduction
Motility iii. grip the plug of the third tube between the ring and middle finger and
Size multicellular Present/ Sexual/ never lay any of the plugs down
organisms absent Asexual d.flame the mouths of the tubes
 e.insert the loop into the culture tube and obtain a loopful of the inoculum (if  beef extract, a hydrolyzed extract of meat at a concentration of 0.3 – 0.5%
broth shake the culture a bit first) or a small amount of growth (if will support the growth of microorganism when combined with peptone
contained in a solid medium). If a mold culture is to be transferred. A  contains peptides and amino acids, nucleotide fractions, organic acids,
small piece of colony together with the agar maybe removed minerals, and some vitamins
f. introduce culture into tube(s) of fresh medium as follows:  its function is to complete the properties of peptone by contributing
i. if the medium is a slant, streak in a zigzag manner from the bottom
minerals, phosphates, energy sources and those essential factors
ii. if the medium is broth, dip the loop in the broth and shake gently to
missing in peptone
dislodge the inoculum
 agar is a component used for solidifying bacteriological media; it is a gel-
iii. if the medium is a stab, insert the loop down in the butt of the agar
forming polysaccharide, which is extractable from several species of red
iv. if more than one tube is to be inoculated, inoculum may again be
seaweeds (agarophytes)
obtained from the culture tube before inoculating the next tube.
(sterilize the loop before getting inoculum again). Work as closely as  a mixture of at least 2 polysaccharides: agarose and agaeopectine, with
possible near the flame components
g.when transfer has been completed, flame the mouths of the tubes and  should be capable of remaining molten and fluid at 40 - 45°C after
return their corresponding plugs cooling from the boiling point
h.sterilize the loop before laying it on the table  grades are available and depending upon brand, the required amount of
agar will vary
 agar slopes or slants are made by allowing liquefied agar to solidify in
slanting position
 media should always be stored in cool and moist environment to prevent
TRANSFERRING INOCULUM FROM TUBE TO PETRI PLATE evaporation
a.heat wire loop
b.while holding the sterilized loop, remove the cotton plug from the mouth of
the tube containing the inoculum by grasping the plug with the little finger
of the right hand DIFFERENT TYPES OF MEDIA
c. flame the mouth of the culture tube, insert the loop into the culture and BASED ON THE PHYSICAL STATE
obtain inoculum SOLID MEDIUM
d.flame the mouth of the culture tube and return plug. Place tube on a test  this medium has a physical structure that allows bacteria to grow in
tube rack physically informative ways, such as in colonies or streak
e.hold Petri plate with the left hand supporting the bottom using the middle,  it also uses solidifying agents such as agar
ring, and small fingers  an example of this is blood agar
f. lift cover, using thumb and forefinger to open the plate SEMI-SOLID MEDIUM
g.introduce inoculum and work as closely as possible near the flame  is used for the determination of bacterial motility, promoting aerobic growth,
POURING MEDIA and fermentation studies
Several precautions are necessary to prevent contamination of the sterile  also contains agar but only in 0.2-0.4% concentration
media. See to it that the outside surface of the media container is dry;  an example of this is Stuart’s Media
otherwise, the liquid clinging to the sides of the container will run into the plate LIQUID MEDIUM
and introduce contaminants  is known for its greater sensitivity of the isolation of small numbers of
a.remove the cotton plug or cap using the little finger of the right hand and microorganisms
flame the mouth of the container. Without putting down the plug, transfer  where bacteria grow uniformly and produces general turbidity
container to the right hand  the following are the example of this media: nutrient broth, sugar media,
b.hold the petri plate or stack plates on the working table and enrichment media
c. pour the agar while raising the cover of the plate only on one side and just BASED ON THE INGREDIENTS
sufficiently, to admit easily the mouth of the container
SIMPLE MEDIUM
d.after pouring, lower the cover into plate immediately
 it contains nutrient broth and peptone water
e.for inoculated plates, gently tilt from side to side or put the plate down and
move it in a back- and-forth, right-left direction to distribute the agar  basis of other media
uniformly over the bottom. Do not let the medium come into contact with  an example of this is nutrient broth
the cover COMPLEX MEDIUM
 it contains mixture of many chemicals in unknown proportions
PIPETTING
 usually contains water, a carbon source, salts, and source of amino acid
a.remove sterile pipette from container. Get the first pipette that is touched.
and nitrogen
Never return pipette that is taken out of the container. Do not let pipette
touch anything  an example of this is blood agar
b.using the pipette aid, draw out the desired amount of sample . (Never use SYNTHETIC OF DEFINED MEDIUM
the mouth to pipe human pathogens or toxin producers like Clostridium).  it is the contrast of complex medium
Control the flow of fluid using the forefinger  all of its ingredients are known
c. flame mouth of sample container, return plug and set aside container  does not contain any animal, yeast, or plant tissue
d.place pipette in receptacle after using. Do not lay it down on the table  consists of trace elements, vitamins, and a carbon and nitrogen source
e.to transfer sample to plate, hold and open the plate. Introduce sample by SPECIAL MEDIA
releasing the forefinger. Do not blow out contents. Otherwise, let tip of a. ENRICHED MEDIA
pipette touch dry area in the plate to deliver the last drop. Work as closely  is used to facilitate the growth of bacteria by adding substances
as possible near the flame like blood, serum, and egg to the basal media in order to meet its
f. to transfer sample to tube, handle tube and pipette nutritional requirements
P R E P A R A T I ON O F C U L T U R E ME D IA  an example of this is chocolate agar
b. ENRICHMENT MEDIA
 culture medium is any material prepared for the cultivation of microorganism
 this is a liquid media that is known to stimulate or suppress the
 must satisfactorily supply all the factors required by the microorganisms for growth of bacteria for the sake of isolation
growth
 an example of this is tetrathionate broth
 chemical compounds may be grouped as: 1. Nitrogen sources; 2. Carbon c. SELECTIVE MEDIA
sources; 3. Energy sources; 4. Minerals; 5. Growth factors; and other
 it is a solid media that contain substance which inhibit
substances maybe added as needed; for example, the buffer salt, indicator
commensalism where growth and isolation of certain bacteria is
dye, reducing substances, and selective agents
expressed
 culture medium can be prepared from basic ingredients or from
 an example of this is Thayer Martin Medium
commercially available digests
d. DIFFERENTIAL OR INDICATOR MEDIA
 most commonly used liquid medium is nutrient broth. However, coconut
 it is designed to easily recognize different bacteria based on their
water can be used as substitutes
colony color
 contains sugars, amino acids, vitamins, and other growth factors  an example of this is Cysteine Lactose Electrolyte Deficient Agar
needed by the organism e. TRANSPORT MEDIA
 with fastidious organism, other compounds maybe added to the nutrient  is used to maintain the viability of certain organisms in clinical
broth or coconut water in order to meet the needs of the specific bacteria specimens during their transport to the laboratory
 when a solid medium is wanted, agar is added  an example of this is Stuart’s Medium
 agar-agar shreds can used as substitute
M IC R OO R G A N IS MS IN T H E E N V IR O N ME N T b. obtain three 9-ml water blanks in screw cap tubes and label each tube
from 1 to 3
 microorganisms, despite of their size are major contributors to the chain of c. secure a broth mix culture
events that can alter the ecology of the environment d. with a sterile pipet, transfer one ml from the stock culture to tube #1.
 an example of this phenomenon is their ability to pollute bodies of water, Cover and shake vigorously
transmit diseases to other forms of living organisms and even synthesize e. from tube #1, transfer 1 ml to tube #2 and mix
products useful to other living forms f. again transfer 1 ml from tube #2 to tube #3, then shake and mix
 they can be found in air, water, soil and even inside the human body g. using a pipet, transfer 1 ml from each tube to a duplicate sterile plate.
 microbes thrives everywhere as long as their physical and nutrient needs Pour melted agar and mix
are met h. allow the agar to solidify and incubate
PROCEDURE i. examine after 48 hours
SPREAD PLATE
a. using a cotton bud, rub the screen of your cellphone and gently swab or  a technique to plate a liquid sample containing bacteria so that the bacteria
streak it on the disposable container that has a label of CELLPHONE are easy to count and isolate
SCREEN and cover PROCEDURE
b. open the disposable container with the label COUGH and cough for 3-5 a. pour melted NA or CWA into each of six sterile petri dishes and allow to
times on it. Cover solidify
c. using a cotton bud, rub the side of the sink and gently swab or streak it on b. transfer to duplicate plates 0.1 ml from each tube used in pour plate
the disposable container that has the label of SINK and cover technique
d. don’t touch the disposable container with the label UNTOUCHED c. sterilize an L-shaped glass rod by dipping in alcohol and flaming until
e. wrap each disposable containers with a paper or aluminum foil and leave alcohol has burned off
in a warm area such as on top of a refrigerator for one week. Use tape to d. spread the inoculum evenly on the surface of the agar with an L-shaped
hold the paper rod
CHARACTERISTICS OF COLONIES OBSERVED
Petri Colonies Size Color Shape Texture
dishes
Cough ~900 1mm-5mm White Round Smooth
in diameter
CP ~500 1mm-7mm White Mostly Round Mostly
Screen in diameter Smooth
Some Irregular
Some
Rough
Sink ~1000 1mm-10mm White Mostly Round Mostly
in diameter Smooth
Some Irregular
Some
Rough
/ ~100 1mm-10mm White Round Smooth
Untouch in diameter
ed

I SO L A T IO N OF MI C R O OR GA N I SM S
 microorganisms in nature are diverse and occur as a complex mixed
population
 study of these microorganisms requires that they be separated from each
other and cultivated in an artificial environment, and this is called isolation
 different techniques are employed to isolate the different species from the
mixture and cultivate them as pure cultures
 pure culture is consists of a population of cells derived from a single cell
 the use of enrichment or selective medium will enhance the growth of the
microorganisms or interest while inhibiting or killing the other types of
microorganisms
DIFFERENT TYPES OF ISOLATION METHOD
STREAK PLATE METHOD
 streak plate is a very simple method of isolation if done properly
 melted nutrient agar is added first, followed by a loop of bacteria from a
slant
 pure colonies develop faster
PROCEDURE
a. pour melted NA or CWA, following aseptic technique in a petri dish and
allow the agar to solidify
b. sterilize the wire loop and get a loopful of microorganisms of interest
from the petri dish with mix culture done
c. streak very gently over the surface of the agar, taking care not to
destroy it
d. incubate the dish invertedly and examine the plate after 48 hours
e. examine your previous petri dish further. Choose another colony and
get a loopful
f. this time streak this in an agar slant
g. incubate and examine tube after 48 hours
POUR PLATE METHOD
 plating method can also be used, and in addition, can approximate the
variable organisms in the population
 bacterial broth is introduced first, followed by the nutrient agar
PROCEDURE
a. liquefy medium in a water bath