Lecture 1

Introduction to Medical Microbiology

Mid-term
1 - microscopic
1- staining
1- ID maldi tof
1- safety
1- pathogenesis

SCI 8007SEF Medical Microbiology & Virology

By Dr. Andy YY CHEUNG

cheungyy@hkmu.edu.hk
 Book references

https://pre-med.jumedicine.com/wp-content/uploads/sites/7/2018/09/Murray-Medical-Microbiology-8th-Edition-c2016.pdf

• Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., &

Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical
Microbiology 26th Ed., by The McGraw-Hill Companies.
https://biologywala.com/wp-content/uploads/2022/08/Jawetz-Melnick-Adelbergs-Medical-Microbiology-27-E-Karen-C.-Carroll-Janet-Butel-Stephen-Morse-biologywala.com_

• Mahon, C. R., & Lehman, D. C., (2019). Textbook of diagnostic

microbiology. Elsevier. http://repository.stikesrspadgs.ac.id/42/1/
Textbook%20of%20Diagnostic%20Microbiology-1060hlm.pdf

1 questions will be included in the mid-term regarding to Pathogenesis in Pre-lectures

 Evolution of Theories of Disease
-Humoral theory & Miasma theory

https://www.youtube.com/watch?v=9e3eOKX3tYw
 Evolution of Theories of Disease
Miasma theory -> Germ theory

https://www.youtube.com/watch?v=N9LC-3ZKiok
 Evolution of Theories of Disease
-Germ Theory of Diseases and Koch’s Postulates

https://www.youtube.com/watch?v=97sEcWEb3Iw
 information only, not included in assessment

Development of microbiology
• Dutch biologist Anton van Leeuwenhoek in 1674 as he peered through his carefully ground microscopic
lenses at a drop of water and discovered a world of millions of tiny “animalcules”
• Almost 100 years later, the Danish biologist Otto Müller extended van Leeuwenhoek’s studies and organized
bacteria into genera and species according to the classification methods of Carolus Linnaeus. This was the
beginning of the taxonomic classification of microbes
• In 1840, the German pathologist Friedrich Henle proposed criteria for proving that microorganisms were
responsible for causing human disease (the “germ theory” of disease)
• Robert Koch and Louis Pasteur confirmed this theory in the 1870s and 1880s with a series of elegant
experiments proving that microorganisms were responsible for causing anthrax, rabies, plague, cholera, and
tuberculosis
• The era of chemotherapy began in 1910, when the German chemist Paul Ehrlich discovered the first
antibacterial agent, arsphenamine (Salvarsan), a compound effective against the spirochete that causes
syphilis
• This was followed by Alexander Fleming’s discovery of penicillin in 1928, Gerhard Domagk’s discovery of
sulfanilamide in 1935, and Selman Waksman’s discovery of streptomycin in 1943
• In 1946, the American microbiologist John Enders was the first to cultivate viruses in cell cultures, leading
the way to the large-scale production of virus cultures for vaccine development

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
Koch’s Postulates and its limitations
As originally stated, the four criteria are:
• (1) The microorganism must be found in diseased but not healthy
individuals;
• (2) The microorganism must be cultured from the diseased individual;
• (3) Inoculation of a healthy individual with the cultured microorganism
must recapitulated the disease; and
• (4) The microorganism must be re-isolated from the inoculated, diseased
individual and matched to the original microorganism
Koch’s postulates have been critically important in establishing the criteria
whereby the scientific community agrees that a microorganism causes a
disease
The germ theory of disease and its limitations
• Germ theory states that specific microscopic organisms are the cause of
specific diseases
• “infectious disease is primarily caused by transmission of an organism from
one host to another—is a gross oversimplification”
• “accords with the basic facts that infection without an organism is
impossible and that transmissible organisms can cause disease”
• “but it does not explain the exceptions and anomalies”
• “The germ theory has become a dogma because it neglects the many other
factors which have a part to play in deciding whether the
host/germ/environment complex is to lead to infection”
• “Among these are susceptibility, genetic constitution, behavior, and
socioeconomic determinants”

Stewart, G. T. (1968). Limitations of the germ theory. The Lancet, 291(7551), 1077-1081.
 Host-Pathogen-Environment-Microbiome
interaction

immunity

Schmeller, D. S., Courchamp, F., & Killeen, G. (2020). Biodiversity loss, emerging pathogens and human health risks. Biodiversity and conservation, 29, 3095-3102.
Development of microbiology
• Our knowledge of microbiology is now undergoing a remarkable transformation founded
in the rapid technologic advances in molecular biotechnology
• The Human Genome Project was a multinational program that concluded in 2005 with
the comprehensive sequencing of the human genome
• The techniques developed for this program have rapidly moved into the research and
clinical laboratories, leading to microbial sequencing and revealing previously
unappreciated insights about pathogenic properties of organisms, taxonomic
relationships, and functional attributes of the endogenous microbial population
• Clearly, we are at the early stages of novel approaches to diagnostics and therapeutics
based on the monitoring and manipulations of this population (the microbiome)
• We now know that there are thousands of different types of microbes that live in, on,
and around us—and hundreds that cause serious human diseases
• To start, the microbes can be subdivided into the following four general groups: viruses,
bacteria, fungi, and parasites, each having its own level of complexity

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
Microscopy
• In general, microscopy is used in microbiology for two basic purposes:
➢ the initial detection of microbes and
➢ the preliminary or definitive identification of microbes
➢ bacterial cells, fungal elements, parasites (eggs, larvae, or adult forms),
and viral inclusions present in infected cells
• For examples, by characteristic morphologic properties:
➢ preliminary identification of most bacteria
➢ definitive identification of many fungi and parasites
• With antibodies labeled with fluorescent dyes or other
➢ specific identification of organisms in clinical specimens

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
Microscopy
• The Light Microscope
• Resolving power: about half the wavelength of the light being used
• Resolving power is the distance that must separate two point sources of light
if they are to be seen as two distinct images.
• With yellow light of a wavelength of 0.4 mm, the smallest separable
diameters are thus about 0.2 mm (i.e. 1/3 width of a typical prokaryotic cell)
• Useful magnification of a microscope is the magnification that makes
visible the smallest resolvable particles.

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Light Microscope

Image source: https://microbenotes.com/light-microscope/

 Observation with a light microscope
(low-power field and high-power field)

https://www.youtube.com/watch?v=QhjT6_4zDws
 Observation with a light microscope
(low-power field and high-power field)
to focus the microscope
- low power
adjust the coarse adjustment

rotate to the next power

use only the fine adjustment

turn to oil lens

before reaching to 100x
drop oil
then 100x
it shd be touching the oil

—> 10x to return

https://www.youtube.com/watch?v=SUo2fHZaZCU
 Observation with a light microscope
(Oil Immersion Microscopy; Oil lens field)

https://www.youtube.com/watch?v=zzamomqlwxU
Several types of light microscopes are
commonly used in microbiology:
• A. Bright-Field Microscope
• consists of two series of lenses (objective and ocular lens), which function
together to resolve the image
• a 100-power objective lens with a 10-power ocular lens magnifies the specimen
1000 times
object smaller then 0.2 um —> cannot be resolved using bright-field
• Particles 0.2 µm in diameter: magnified to about 0.2 mm and so become visible
Further magnification: give no greater resolution of detail and
reduce the visible area (field)
• With this microscope, specimens are rendered visible because of the differences
in contrast between them and the surrounding medium.
• Bacteria are difficult to see: lack of color and contrast with the surrounding
medium
• Dyes (stains): increase the contrast, bacteria becomes more easily seen
Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Several types of light microscopes are
commonly used in microbiology:
• B. Dark-Field Microscope
• lighting system modified to reach the specimen from the sides only
• accomplished by the use of a special condenser that both blocks direct
light rays and deflects light off a mirror on the side of the condenser at an
oblique angle
• creates a “dark field” that contrasts against the highlighted edge of the
specimens and results when the oblique rays are reflected from the edge of
the specimen upward into the objective of the microscope
➢Higher resolution
• useful to observe organisms that is smaller than 0.2 µm in diameter e.g.
Treponema pallidum , a spirochete
Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Image source: https://microscopewiki.com/brightfield-vs-darkfield-microscope/
 Darkfield Microscopy Tutorial
condenser aperture number should be larger than that of the objective lens used.
If the objective aperture number is larger, dark-field cannot be achieved because direct light will be transmitted.

https://www.youtube.com/watch?v=Tw557w421is
Treponema pallidum by Dark-Field Microscope

https://phil.cdc.gov/details.aspx?pid=2335
Several types of light microscopes are
commonly used in microbiology:
• C. Fluorescence Microscope
• The fluorescence microscope is used to visualize specimens that fluoresce , which is the ability to
absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible)
• Some organisms fluoresce naturally because of naturally fluorescent substances e.g. chlorophyll
• Those that do not naturally fluoresce may be stained with fluorochrome (fluorescent dye)
• Fluorescence microscopy is widely used in clinical diagnostic microbiology.
• Fluorescent-antibody (FA) technique or immunofluorescence.
• For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light,
is strongly absorbed by Mycobacterium tuberculosis, the bacterium that causes tuberculosis.
When the dye is applied to a specimen suspected of containing M tuberculosis and exposed to
ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms
against a dark background.
• The fluorescent treponemal antibody absorption (FTA-ABS) test

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
 Diagnosis of active tuberculosis disease:
From microscopy to molecular techniques
fluorescence with better contrast
than acid fast for MTB

https://phil.cdc.gov/PHIL_Images/20040
615/238aba2af0c347f7a0f744ff52b7c10 https://phil.cdc.gov/PHIL_Images/20041202/1a
d/5790_lores.jpg 0f953285f84cd4b3d1b31237a7a5a8/6468_lores
.jpg

Caulfield, A. J., & Wengenack, N. L. (2016). Diagnosis of active tuberculosis disease: From microscopy to molecular techniques. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, 4, 33-
43.
Fluorescent treponemal antibody absorption
(FTA-ABS) test

Image source:
https://microbeonline.com/fluorescent-treponemal-antibody-absorption-fta-abs-test/
Besides light microscopes, electron
microscopes are also used in microbiology:
** NOT electronic microscope
can be used for observing virus
D. Electron Microscope
• High resolving power: observe the detailed structures of microorganisms
• Superior resolution of the electron microscope is due to the fact that electrons have a much shorter
wavelength than the photons of white light —> can observe microorganisms with diameter smaller than 0.2 um
• Two types of electron microscopes: the transmission electron microscope (TEM), which has many features
in common with the light microscope, and the scanning electron microscope (SEM)
• TEM
➢ uses a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser
lens onto a thin specimen
➢ electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen
➢ some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens,
which presents an image of the specimen to the projector lens system for further enlargement
➢ When the electrons struck a screen, it fluoresces and image is visualized
➢ Image visualized by a fluorescent screen, a layer of photographic film, or a sensor attached to a charge-coupled
device
➢ TEM can resolve particles 0.001 µm apart; Viruses with diameters of 0.01–0.2 µm can be resolved
light microscope only resolve 0.2 um

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
 x structure
v how to use
Transmission Electron Microscope v when to use it
v functions

Image source: Image source:

https://www.cambridge.org/core/books/analytical-geomicrobiology/applications-of-transmission- https://www.hitachi-hightech.com/global/science/products/microscopes/electron-
electron-microscopy-in-geomicrobiology/04F139754543AF817BAEAC6DB9608B29 microscope/tem/ht7800.html
Virus size comparison

https://viralzone.expasy.org/5216 Can you see a virus with the light microscope?

No, except poxviruses. the largest virus, but still difficult to be seen
 Monkeypox virus taken by light microscope

hematoxylin-eosin, original magnification, immunoperoxidase, original magnification,

× 25 [A] × 150 [B]

immunoperoxidase, original magnification, immunoperoxidase, original magnification,

× 25 [C] × 10 [D]

hematoxylin-eosin, original magnification, immunoperoxidase, original magnification,

× 150 [E] × 150 [F]

immunoperoxidase, original magnification, immunoperoxidase, original magnification,

× 150 [G] × 150 [H]

Zaucha, G. M., Jahrling, P. B., Geisbert, T. W., Swearengen, J. R., & Hensley, L. (2001). The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca
fascicularis). Laboratory investigation, 81(12), 1581-1600.
Microscopy- Direct Examination
Direct examination methods are the simplest for preparing samples for
microscopic examination
usually for stool parasite, so that parasite can still survive
➢Wet mount: suspended in water or saline
➢KOH method: mixed with alkali (e.g. potassium hydroxide) to dissolve
background material human specimens e.g. skin —> observe fungal filament
➢Or, mixed with a combination of alkali and a contrasting dyefor a better contrast
• The dyes nonspecifically stain the cellular material, increasing the contrast
with the background, and permit examination of the detailed structures
➢India ink method: ink darkens the background rather than the cell
➢to detect capsules surrounding organisms, such as the yeast Cryptococcus and
encapsulated Bacillus anthracis

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
Microscopy- Direct Examination
the organisms are not killed, they can move around in wet mount
—> used to observe with structure that are easily destroyed by heat fixed / dye
• Wet mount of Trichomonas vaginalis
https://www.youtube.com/watch?v=rim2dXF3Oac
• KOH test of fungi
• India ink method dark background - ink

e.g.
dip infected nail in KOH
dissolved and release the fungi

capsules are not dyed

Image source: https://microbeonline.com/cryptococcus- Image source: https://universe84a.com/koh-test/
neoformans-properties-pathogenesis-diseases-lab-diagnosis/
Microscopy- Differential Stains
A variety of differential stains are used to stain specific organisms or
components of cellular material
• Gram stain: *practical:
perform gram stain
• best known and most widely used stain differentiate gram pos and gram neg
• Bacteria and yeasts (yeasts are gram-positive)
• Acid fast stain
• Differentiate acid fast bacteria (e.g. mycobacterium) from non-acid fast
bacteria
• Iron hematoxylin and trichrome stains: protozoan parasites
• Wright-Giemsa stain: blood parasites

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
Gram staining technique draw circle on the slide using wax pencil (won’t be removed) on the
opposite side
sterilise the loop
transfer a loop of sterile water/ saline on the circle
sterilise another loop and cool down (touch the agar surface to see)
transfer a colony to the circle (emulsify it with the water)
spread to form a thin film
sterilise the loop again
allow the liquid to evaporate (air-dry)
heat fix by flaming above bunsen burner the using the slide holder
crystal violet stain one min
dipping it in water until stain removed (usually used in H&C)
iodine stand one min
rinse
add decolorizer on the slide
rinse
safranin one min
rinse

5 mins to complete two slide in practical

https://www.youtube.com/watch?v=sxa46xKfIOY
 Gram staining technique
used for differentiate (more than one stain is used) gram pos and gram neg
due to different cell wall composition

crystal violet —> give both gram bacteria color

iodine —> fixation

methanol / —> gram neg are decolorised since it only has a thin peptidoglycan layer and high lipid content,

safranin

blot dry

fix the smear on the staining stack

water bottle is used to rinse the smear after staining
after all washing, use blotting water to dry

observe with oil lens immersion (100x)

**report with morphology**, arrangement not required

e.g. gram positive cocci with cluster arrangement/ arrange in chain
gram negative bacilli/rod with no specific arrangement
*All steps for 30 sec in HKMU
https://www.youtube.com/watch?v=--OvDvS-Pec
Microscopy- Differential Stains
A variety of differential stains are used to stain specific organisms or
components of cellular material
• Gram stain:
• best known and most widely used stain
• Bacteria and yeasts (yeasts are gram-positive)
• Acid fast stain
• Differentiate acid fast bacteria (e.g. mycobacterium) from non-acid fast
bacteria
• Iron hematoxylin and trichrome stains: protozoan parasites
• Wright-Giemsa stain: blood parasites

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
 Iron hematoxylin staining

trichrome staining
Walochnik, J., & Aspöck, H. (2012). Protozoan pathogens: identification. Els.

Image source:
Image source: https://microbeonline.com/trichrome-
https://www.quora.com/What-are-the-mechanisms-
staining-for-fecal-smears/
of-gram-staining-and-how-do-they-work

malaria
Wright-Giemsa

Image source:
https://www.dreamstime.com/malaria-parasite-red-blood-cells-ring-form-stage-plasmodium-falciparum-
original-magnification-x-image165229628
 Microscopy- Acid-Fast Stains
• Ziehl-Neelsen staining:
➢oldest method, requires heating the specimen during staining
• Kinyoun method:
➢cold acid-fast stain
• fluorochrome stain (auramine-rhodamine) used for first screen for MTB in HA/DH
Fluorochrome method: a large area of the specimen can be examined rapidly by
simply searching for fluorescing organisms against a black background
software is able to differentiate fluorescence
once detected
differentiate if it is MTB
v no need to depend on naked eyes

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
 Diagnosis of active tuberculosis disease:
From microscopy to molecular techniques
maybe hard to differentiate easily used to distinguish MTB

https://phil.cdc.gov/PHIL_Images/20040
615/238aba2af0c347f7a0f744ff52b7c10 https://phil.cdc.gov/PHIL_Images/20041202/1a
d/5790_lores.jpg 0f953285f84cd4b3d1b31237a7a5a8/6468_lores
.jpg

Caulfield, A. J., & Wengenack, N. L. (2016). Diagnosis of active tuberculosis disease: From microscopy to molecular techniques. Journal of Clinical Tuberculosis and Other Mycobacterial Diseases, 4, 33-
43.
 Microscopic examination
under 100x (oil)
report with positive for acid fast bacteria with xx arrangement
Acid-fast staining
differentiate acid fast bacteria
Ziehl-Neelson technique

acid fast bacteria: difficult to be stain due to mycolic acid which a waxy material around the
surface, but once stained with strong carbon fuchsin by heat treatment and applied with acid-
alcohol, they will not be decolorised,

then methylene blue added to stain non-acid fast bacteria

/ malachite green
steps:
air dry and heat fix the smear first (performed in BSC since MTB is airborne pathogens)

cover the slide with strong carbon fuchsin and

heat the stain under the slide for 8 mins so that stain and go inside

cool down and rinse with distilled water

cover the slide with acid-alcohol 2 min/ until slide decolorised to pale pink

rinse

cover with methylene blue 30 sec to 1 min

rinse
https://www.youtube.com/watch?v=x2sXdMbJoNk
blot dry
Microscopy- Fluorescent Stains
For example,
• Auramine-rhodamine: Acid-fast stain
• Acridine orange stain: bacteria and fungi
• limited application
• Calcofluor white stain: chitin in fungal cell walls
• replaced the potassium hydroxide stains
• Fluorescent antibody stain
Image source:
Ab binds to target, Ab stain bind to Ab https://biotium.com/product/calcofluor-white-stain-5-mm-water/

Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition.
 Auramine staining for tuberculosis diagnosis
QC required in staining

https://www.youtube.com/watch?v=JjuYAL4Hqho
Lecture 1
Introduction to Medical Microbiology
-supplementary slides
usually used in research field,
hardly seen in routine lab

SCI 8007SEF Medical Microbiology & Virology

By Dr. Andy YY CHEUNG

cheungyy@hkmu.edu.hk
Several types of light microscopes are NOT
commonly used in microbiology:
• Phase Contrast Microscope (NOT commonly used in routine)
• Light waves passing through transparent objects, such as cells, emerge in
different phases depending on the properties of the materials through
which they pass
• This effect is amplified by a special ring in the objective lens of a phase
contrast microscope, leading to the formation of a dark image on a light
background
• improve contrast differences between cells and the surrounding medium
• see living cells without staining them;
(with bright-field microscopes, bacteria is killed and stained)
Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Image source: https://microbeonline.com/phase-contrast-microscope/ Image source: https://www.olympus-lifescience.com/en/microscope-
resource/primer/techniques/phasecontrast/phase/
Several types of light microscopes are NOT
commonly used in microbiology:
• Differential Interference Contrast Microscope (NOT commonly used in routine)
• Differential interference contrast (DIC) microscopes employ a polarizer to
produce polarized light
• The polarized light beam passes through a prism that generates two distinct
beams; these beams pass through the specimen and enter the objective lens,
where they are recombined into a single beam
• Because of slight differences in refractive index of the substances each beam
passed through, the combined beams are not totally in phase but instead create
an interference effect, which intensifies subtle differences in cell structure
• Structures such as spores, vacuoles, and granules appear three-dimensional
• DIC microscopy is particularly useful for observing unstained cells because of its
ability to generate images that reveal internal cell structures that are less
apparent by bright-field techniques
Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
 Differential Interference Contrast Microscope

Image source: Image source:

https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/dic/dicintro/ https://canadiannaturephotographer.com/diffential_interference_microscopy.html
Several types of light microscopes are NOT
commonly used in microbiology:
Scanning electron microscope (SEM) (NOT commonly used in routine)
• generally lower resolving power than TEM
• useful for providing three-dimensional images of the surface of microscopic objects
• Electrons are focused by means of lenses into a very fine point
• The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from
the surface of the material, which can be captured by an appropriate detector, amplified, and then imaged
• “Shadowing” in EM:
• Deposite a thin layer of heavy metal on the specimen by placing it in the path of a beam of metal ions in a vacuum
• The beam is directed at a low angle to the specimen so that it acquires a “shadow” in the form of an uncoated area on the
other side
• When an electron beam is then passed through the coated preparation in the electron microscope and a positive print is
made from the “negative” image, a three-dimensional effect is achieved
• Use of ultrathin sections of embedded material
• a method of freeze-drying specimens that prevents the distortion caused by conventional drying procedures, and
• the use of negative staining with an electron-dense material such as phosphotungstic acid or uranyl salts
• Without these heavy metal salts, there would not be enough contrast to detect the details of the specimen
Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Scanning electron microscope

Image source: https://www.thermofisher.com/blog/materials/what-is-sem-scanning-electron-microscopy-explained/

Several types of light microscopes are NOT
commonly used in microbiology:
Confocal Scanning Laser Microscope (NOT commonly used in routine)
• couples a laser light source to a light microscope
• a laser beam is bounced off a mirror that directs the beam through a scanning device
• Then the laser beam is directed through a pinhole that precisely adjusts the plane of focus of the beam to a
given vertical layer within the specimen
• By precisely illuminating only a single plane of the specimen, illumination intensity drops off rapidly above
and below the plane of focus, and stray light from other planes of focus are minimized
• Thus, in a relatively thick specimen, various layers can be observed by adjusting the plane of focus of the
laser beam
• Cells are often stained with fluorescent dyes to make them more visible.
• Alternatively, false color images can be generated by adjusting the microscope in such a way as to make
different layers take on different colors
• equipped with computer software to assemble digital images for subsequent image processing.
• Thus, images obtained from different layers can be stored and then digitally overlaid to reconstruct a three-
dimensional image of the entire specimen

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Confocal Scanning Laser Microscope

Image source: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/confocal/confocalintro/

Several types of light microscopes are NOT
commonly used in microbiology:
Scanning Probe Microscopes (NOT commonly used in routine)
• A new class:
• measure surface features by moving a sharp probe over the object’s surface
• Examples:
• scanning tunneling microscope and atomic force microscope
• view atoms or molecules on the surface of a specimen
• E.g. interactions between proteins of the bacterium Escherichia coli

Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies.
Lecture 1
Introduction to Medical Microbiology
-Laboratory technique

SCI 8007SEF Medical Microbiology & Virology

By Dr. Andy YY CHEUNG

cheungyy@hkmu.edu.hk
avoid streaking too close to the perimeter because bacteria may grow along the side —> change the entire growing environment
(since some plate change colour with pH change)

Four Quadrant Streak procedure

Text

https://www.youtube.com/watch?v=FutAgWDymAM
Streak plate technique
4 quadrants
Streak plate technique

5 quadrants performed
Streak plate technique
first done by cotton swab xxx
loop should be used

streaking back to the first quadrants

Streak plate technique

streak too close to the perimeter

Pour Plating (not commonly used in routine
practice)

https://www.youtube.com/watch?v=0X39eeZbS98
Spread Plate Technique for Colony Counting
(not commonly used in routine practice)

https://www.youtube.com/watch?v=gwPZhsyMI9M
 Differentiate tests

How to Perform the Catalase Test -

Staphylococcus vs Streptococcus
Both dark blue in gram stain and cocci
Staph positive
Strep negative

3% H2O2 required

loop to transfer one colony to slide

use dropper 2-3 drops H2O2 on the colony without mixing

Negative: no bubbling effect

Positive: bubbles observed since H2O2 are broken down

https://www.youtube.com/watch?v=krHqynVe6-g
 Staphaurex test for Staphylococcus aureus
a black background required

a loop of water by a loop to the slide

take a loop of bacteria inside the water on the slide and mix well resulting in milky

add a drop of staphaurex suspension, add in vertical angle

rock slide and look for clumping

clumping = Stap. aureus

*noted that there are some Straphylococcus spp. show positive results
<1% false positive
so some lab will perform further test like MALDI TOF

https://www.youtube.com/watch?v=ntn7zOOw9-Q
 All differential test require QC
coagulase in Stap aureus —> clot / clumps

Slide and tube coagulase tests

for Stap. aureus (positive); Stap spp. (negative)
Slide: divide the slide into two parts using waz pen and draw two circles
place a drop of saline into the circle
transfer and emulsify 1-2 colonies on saline (shd look milky)

Clumps observed: positive

look smooth and milky: negative

Tube: pick 1-2 colonies and put inside the tube with reagent

clot can be observed by sliding the tube

https://www.youtube.com/watch?v=Mtxs9iagW40
Rabbit coagulase plasma (Coagulase test)
beta:
TSA Sheep's Blood Medium - Hemolysis
complete break down of RBCs
(can see through the agar,
transparent)

alpha:
greenish color, partially break down
blood

Gamma:
shiny white growth
no break down of blood

https://www.youtube.com/watch?v=-ysnHBMToBo
 Bile solubility test
Streptococcus pneumoniae vs other alpha hemolytic Streptococcus

- direct method
directly add bile salt on suspected colony
after incubation, colony lysed by bile salt —> clear zone

- broth culture method

pick up suspected colony and inoculate into the broth
after incubation, bile salt solution added
Strep pneumoniae will be lysed by bile salt
and become clear

https://www.youtube.com/watch?v=fvB9FUTFUl4
 How to perform an indole test by a direct
swab or a spot indole test for E.coli (one of the bacteria)

filter paper on the petri dish flooded with indole reagent

—> place the picked colony on the filter paper with swab

https://www.youtube.com/watch?v=rIGfr_v9h2o
https://www.youtube.com/shorts/7WndCnE3kfY
 Microbiology: Indole Test by SIM testSulphide Indole Motility
for gram neg bacilli

theory:
bacteria expressing
tryptophanase that can
hydrolyse tryptophan to
indole.

Kovac’s reagent react

with Indole* to produce
red color on the top
= positive
bacteria do expressed
trytophanase

https://www.youtube.com/watch?v=m27wzevB9bA
 To differentiate members of Enterobacteriaceae
the medium have ferrous ammonium sulfate, sodium thiosulfate —> serve as an indicator for hydrogen sulfide H2S production
==> a black precipitate detected

Motility test
hold the inoculation loop with colonies and stab it straight down to the center,
then remove the loop along the stab line
Negative
Positive: a diffuse zone of growth flaring for
from the line of inoculation motility
/ fuzzy area around the stab line

Negative: restricted growth,

indicated by no bacterial growth
along the stab line
 How to perform an Oxidase test in
microbiology Pseudomonas, aeromonas, Neisseria, Vibrios,
Brucella, Campylobacter, Pasteurella

https://www.youtube.com/watch?v=c5sRWt3DKVw
for detecting the cytochrome oxidase activity in bacteria
initiation characterisation of gram negative bacteria

oxidase oxidises the TMPD reagent to indphenol which is a dark blue end product

The media used from obtaining colonies must be non-selective or non-differential, contains no dye
because dyes may produce error, media with high carbohydrates should not be used.

Put a filter paper on a petri dish and moisten it with a few drops of oxidase agent

use a loop to transfer a loop of bacteria and put it on the filter paper for results

Positive: blue colour can be observed

 for identification of Streptococcus pneumoniae (from other alpha-hemolytic bacteria)

Optochin is a chemical that is toxic to some bacteria

and this test can be used to determine whether the bacteria is susceptible or resistant to optochin
Optochin test Positive: a zone of inhibition (14mm greater)
(If the zone is a bit small, identify further with bile salt to confirm if it is S.pneumoniae)

well defined zone of inhibition results around the impregnated disk:

a positive *presumptive identification of S. pneumoniae (shd be tested with further biochemical test for confirmation)

Other alpha-hemolytic streptococci do not display this clear zone of inhibition when in the presence of optochin. The chemical
tests the fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes in surface tension.
https://microbiologyinfo.com/optochin-susceptibility-test-for-the-identification-of-streptococcus-pneumoniae/
 0.04 units bacitracin uses for presumptive
identification of Streptococcus pyogenes
Bacitracin is an antibiotics that inhibit the synthesis of cell wall,
it will diffuse into the medium and inhibit the growth of susceptible
microorganisms.

Steps:
streak 2-3 suspected colonies onto the agar plate by loop
then incubate to see szone of exhibition other beta-hemolytic streptococci (resistant)
staphylococci species (resistant)

Streptococcus pyogenes (susceptible)

micrococci (susceptible)

histogene.co susceptibility
 10 units for screening Haemophilus from
sputum specimen
Haemophilus:
- Oxidase test positive
- Gram negative bacillus/ coccobacilli

10U bacitracin
(Bacitracin can block cell wall formation by
interfering the dephosphorylation of lipid
compound that carries peptidoglycans to
the growing microbial cell wall)

Haemophilus is resistant to 10U bacitracin

while most common bacteria are sensitive

https://universe84a.com/bacitracin-test/
 Bacitracin Test & Optochin test

https://microbeonline.com/bacitracin-test-principle-procedure-expected-results-and-quality-control/#google_vignette
 used for identifying Streptococcus pyogenes from other beta-hemolytic streoptococci, as well as enterococcus species

- put the disc on a slide and moisten it with distilled water

PYR test - transfer colonies of bacteria to be tested using a loop and smear it on the disc, wait for 2 mins
- add one drop of chromogenic solution
color shd develop within one mins

• PYR is a rapid method for presumptive identification of bacteria based on the pyrrolidonyl
arylamidase enzyme
• The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl- β-naphthylamide substrate
to produce a β-naphthylamine
• The β-naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde
reagent by the production of a bright red precipitate

https://www.youtube.com/watch?v=MNUQSZpI-aA