Exercise 4

SEEING THE
INVISIBLE: USE OF
MICROSCOPE
ALMOITE, BILLONES, LODIA, TAPE
Introduction
Since microbes are very small, we need a
01 microscope to see them.

02 A microscope can reveal from morphological

to ultrastructural details of a cell.

03 The most common is the compound microscope

which has two lenses (objective lens, eyepiece).

04 Objective lens = real image; eyepiece = virtual

05 Objective lenses: Scanner, LPO, HPO, OIO

Introduction
OIO uses oil, has a refractive index like a glass,
06 conserves light, and allows a clearer view of the
specimen.

A regular compound light microscope produces

07 a brightfield illumination thru a condenser which
is used to view unstained cells for motility.

08 Cells can be stained to increase contrast.

It can be used to count and estimate the number

09 of microbes thru a Petroff-Hausser counting
chamber or a hemocytometer.
Materials and Methods
Preparation Techniques of Specimen
and Microscopic Cell Counting
Brightfield
Microscope
 Temporary Wet Mount
Saccharomyces cerevisiae

Place a drop of
Lactophenol Blue Carefully Mix

1 2 3 4

Clean glass slide Aseptically obtain

sample from yeast
culture
 Temporary Wet Mount
Saccharomyces cerevisiae

Observe under LPO

5 6 7

Place coverslip Record observations

diagonally
 Hanging Drop Technique
Pond Water/ Hay Infusion

Place a loopful of the Observe under HPO

sample on a coverslip Record Observations

1 2 3 4

Apply petroleum jelly Place depression slide

on the depression slide over the coverslip and
quickly turn the slide
 Slide CultureTechnique
Rhizopus spp.

Aseptically obtain Incubate for 2-3 days,

sample from mold Observe under scanner,
culture LPO, and HPO

1 2 3 4

Cut agar block Poke the sides of the

agar block and place
coverslip
Hemocytometer
 Preparation of Yeast Suspension
Saccharomyces cerevisiae

Introduce 10µL of yeast

Drain Supernatant, add suspension into the
5mL distilled water to Chamber. View under
cell pellet LPO and count cells

1 2 3 4

Centrifuge 8mL of Yeast Transfer 10µL of yeast

culture suspension and 90µL of
0.4% trypan blue solution
into microcentrifuge tube
RESULTS
Temporary wet mount
• Simple technique
• Good for viewing living organisms
• Detect motility
• Limited time (water dries up quickly)
• Provides sufficient information
• Can be stained
• Also shows cell pattern and cell shape
Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4
Yeast cells in temporary Yeast cells in temporary Yeast cells in temporary Yeast cells in temporary
wet mount (Group 1) wet mount (Group 2) wet mount (Group 3) wet mount (Group 4)
Hanging Drop technique
• Complex technique
• Often used in dark illumination
• Observe motility of bacteria
• Used in long observations
• Reliable in observing motility
• Also shows cell pattern and cell shape
Amoeba Euglena Paramecium Diatoms
Diatoms Paramecium Paramecium
Amoeba
Motility
True motility True motility True motility No movement
No movement True motility True motility

True motility
Motility
True Motility
Observation of
Rhizopus culture
Observations

Stolons
Sporangia

Rhizopus stolonifer under scanner Photo courtesy of: Group 3 (Salvacion,

objective (40X) Carbonilla, Royeca)
Sporangia

Sporangiopores

Rhizopus stolonifer under low power Photo courtesy of: Group 3 (Salvacion,
objective (100X) Carbonilla, Royeca)
Sporangium Sporangiopore

Sporangiospores Hyphae

Rhizopus stolonifer under high power Photo courtesy of: Group 3 (Salvacion,
objective (400X) Carbonilla, Royeca)
Counting of yeast
cells
White cells Viable
Blue cells Non-viable

S. cerevisiae in a hemocytometer under

high power objective (400X)
 Table 2. Number of viable yeast cells per ml
Large corner square Viable cell
Chamber
1 2 3 4 Total count/ml

1 1,905 743 1,789 1,295 5,732 1.4x108

2 1,097 1,103 1,121 1,205 4,500 1.1x108

AVERAGE 1.3x108

Working formula:
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
Viable cell count (live cells per mL) = x DF x CF
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑎𝑟𝑔𝑒 𝑐𝑜𝑟𝑛𝑒𝑟 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
Chamber 1: 5,732
Viable cell count (live cells per mL) = 4
x 10 x 10,000 = 1.4x108

Chamber 2: 4,500
Viable cell count (live cells per mL) = x 10 x 10,000 = 1.1x108
4
 Table 3. Number of non-viable yeast cells per ml
Large corner square Non-viable
Chamber
1 2 3 4 Total cell count/ml

1 38 50 21 135 240 6.1x106

2 138 482 523 73 1,200 3.0x107

AVERAGE 1.8x107

Working formula:
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
Non-viable cell count (Dead cells per mL) = x DF x CF
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑎𝑟𝑔𝑒 𝑐𝑜𝑟𝑛𝑒𝑟 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
Chamber 1: 240
Non-Viable cell count (Dead cells per mL) = x 10 x 10,000 = 6.1x106
4
Chamber 2:
1,200
Non-Viable cell count (Dead cells per mL) = x 10 x 10,000 = 3.0x107
4
 Table 4. Percentage viability of yeast culture
Total number of viable Total number of yeast
Chamber %viability
cells cells

1 5,732 5,972 96%

2 4,500 5,700 79%

AVERAGE 87.5%

Working formula: 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠

Percentage viability = x 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠

5,732
Percentage viability (chamber 1) = 5,972 x 100 = 96%

4,500
Percentage viability (chamber 2) = 5,700 x 100 = 79%
 Review Question
7. Enumerate the advantages and disadvantages of direct cell count in estimating
the number of microbial cells.
Advantages
• Direct counting methods are used to determine bacterial
concentration without the need for advanced equipment.
• Direct counting methods are easy to perform
Disadvantages
• are often slower than other methods.
• need to be heavily diluted prior to plating.
• inaccurate since it is only an estimation of bacterial count
in a certain amount of volume.
Source: Lumen (n.d.), Boundless Microbiology: Counting
Bacteria. Retrieved: September 5, 2019 from
https://courses.lumenlearning.com/boundless-
microbiology/chapter/counting-bacteria/