BACTERIOLOGY

(Methods of Studying Bacteria)

PRELIM

Anne Lorraine Magarette Dulay

MLS 3.1
JULY 3, 2018
Microscopic

Microorganisms are very diverse. They include bacteria, fungi, algae, and protozoa;
microscopic plants (green algae); and animals such as rotifers and planarians. Most
microorganisms are unicellular (single-celled), but this is not universal.

Single-celled microorganisms were the first forms of life to develop on earth,

approximately 3 billion–4 billion years ago. Further evolution was slow, and for about 3
billion years in the Precambrian eon, all organisms were microscopic. So, for most of the
history of life on earth the only forms of life were microorganisms. Bacteria, algae, and
fungi have been identified in amber that is 220 million years old, which shows that the
morphology of microorganisms has changed little since the Triassic period. When at the
end of the 19thcentury information began to accumulate about the diversity within the
bacterial world, scientists started to include the bacteria in phylogenetic schemes to
explain how life on earth may have developed. Some of the early phylogenetic trees of
the prokaryote world were morphology-based. Others were based on the then-current
ideas on the presumed conditions on our planet at the time that life first developed.

Microorganisms tend to have a relatively rapid evolution. Most microorganisms can

reproduce rapidly, and microbes such as bacteria can also freely exchange genes
through conjugation, transformation, and transduction, even between widely-divergent
species. This horizontal gene transfer, coupled with a high mutation rate and many other
means of genetic variation, allows microorganisms to swiftly evolve (via natural
selection) to survive in new environments and respond to environmental stresses.

The relationship between the three domains (Bacteria, Archaea, and Eukaryota) is of
central importance for understanding the origin of life. Most of the metabolic pathways,
which comprise the majority of an organism’s genes, are common between Archaea and
Bacteria, while most genes involved in genome expression are common between
Archaea and Eukarya. Within prokaryotes, archaeal cell structure is most similar to that
of Gram-positive bacteria.
Cultural
Classification of culture media used in Microbiology laboratory on the basis of
consistency
1. Solid medium
solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly
inert solidifying agent. Solid medium has physical structure and allows bacteria
to grow in physically informative or useful ways (e.g. as colonies or in
streaks). Solid medium is useful for isolating bacteria or for determining the
colony characteristics of the isolate.
2. Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft
custard like consistency and are useful for the cultivation of microaerophilic
bacteria or for determination of bacterial motility.
3. Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of
gelling agents such as gelatin or agar. Broth medium serves various purposes
such as propagation of large number of organisms, fermentation studies, and
various other tests. e.g. sugar fermentation tests, MR-VR broth.
Classification of culture media based on the basis of composition
1. Synthetic or chemically defined medium
A chemically defined medium is one prepared from purified ingredients and
therefore whose exact composition is known.
2. Non synthetic or chemically undefined medium
Non-synthetic medium contains at least one component that is neither purified
nor completely characterized nor even completely consistent from batch to batch.
Often these are partially digested proteins from various organism sources.
Nutrient broth, for example, is derived from cultures of yeasts.
Classification of Bacterial Culture Media based on the basis of purpose/ functional
use/ application

Many special purpose media are needed to facilitate recognition, enumeration, and
isolation of certain types of bacteria. To meet these needs, numerous media are
available.

1. General purpose media/ Basic media

Basal media are basically simple media that supports most non-fastidious bacteria.
Peptone water, nutrient broth and nutrient agar are considered as basal medium. These
media are generally used for the primary isolation of microorganisms.

2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium
makes them enriched media. Enriched media are used to grow nutritionally exacting
(fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of
the enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a
blood agar base. Chocolate agar is also known as heated blood agar or lysed blood
agar.

3. Selective and enrichment media are designed to inhibit unwanted commensal or

contaminating bacteria and help to recover pathogen from a mixture of bacteria. While
selective media are agar based, enrichment media are liquid in consistency. Both these
media serve the same purpose. Any agar media can be made selective by addition of
certain inhibitory agents that don’t affect the pathogen of interest. Various approaches to
make a medium selective include addition of antibiotics, dyes, chemicals, alteration of
pH or a combination of these.

a. Selective medium

Principle: Differential growth suppression

Selective medium is designed to suppress the growth of some microorganisms while
allowing the growth of others. Selective medium are agar based (solid) medium so that
individual colonies may be isolated.

Examples of selective media include:

1. Thayer Martin Agar used to recover N.gonorrhoeae contains antibiotics;

vancomycin, colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10%
NaCl.
3. Potassium tellurite medium used to recover C.diphtheriae contains 0.04%
potassium tellurite.
4. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that
inhibits most gram positive bacteria.
5. Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains
cetrimide (antiseptic agent).
6. Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal
violet.
7. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by
incorporating malachite green.
8. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the
addition of dye brilliant green.
9. Selective media such as TCBS Agar used for isolating V. cholerae from fecal
specimens have elevated pH (8.5-8.6), which inhibits most other bacteria.

b. Enrichment culture medium

Enrichment medium is used to increase the relative concentration of certain
microorganisms in the culture prior to plating on solid selective medium. Unlike selective
media, enrichment culture is typically used as broth medium. Enrichment media are
liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F
broth, tetrathionate broth and alkaline peptone water (APW) are used to recover
pathogens from fecal specimens.

4. Differential/ indicator medium: differential appearance:

Certain media are designed in such a way that different bacteria can be recognized on
the basis of their colony colour. Various approaches include incorporation of dyes,
metabolic substrates etc, so that those bacteria that utilize them appear as differently
coloured colonies. Such media are called differential media or indicator media.
Differential media allow the growth of more than one microorganism of interest but with
morphologically distinguishable colonies.
Examples of differential media include:

1. Mannitol salts agar (mannitol fermentation = yellow)

2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose
fermenter produces pale or colorless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)

5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to
prevent overgrowth of contaminating organisms or commensals. This can be achieved
by using transport media. Such media prevent drying (desiccation) of specimen,
maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria.
Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of
charcoal serves to neutralize inhibitory factors.

 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are
used to transport feces from suspected cholera patients.
 Sach’s buffered glycerol saline is used to transport feces from patients suspected
to be suffering from bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.

6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen
content, reduced oxidation –reduction potential and extra nutrients.

Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin
K. Such media may also have to be reduced by physical or chemical means. Boiling the
medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1%
thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a
medium reduced. Before use the medium must be boiled in water bath to expel any
dissolved oxygen and then sealed with sterile liquid paraffin.

Robertson Cooked Meat (RCM) medium that is commonly used to

grow Clostridium spps contains a 2.5 cm column of bullock heart meat and 15 ml of
nutrient broth. Thioglycollate broth contains sodium thioglycollate, glucose, cystine,
yeast extract and casein hydrolysate.

Methylene blue or resazurin is an oxidation-reduction potential indicator that is

incorporated in the medium. Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g.
antibiotic assay media are used for determining antibiotic potency by the microbiological
assay technique.
Other types of medium includes

 Media for enumeration of Bacteria,

 Media for characterization of Bacteria,
 Maintenance media etc

.
Serological
is defined as, any of several laboratory procedures carried out on a sample of
blood serum, the clear liquid that separates from the blood when it is allowed to
clot. The purpose of such test is to detect serum antibodies or antibody-like
substances that appear specifically in association with certain diseases.
Bacterial Serotyping is an essential process in identifying and distinction of a
certain organism from those with similar characteristics.
a. Lancefield classification- Lancefield grouping is a serological test for
classifying streptococci into one of 20 groups based on the presence
of polysaccharide and teichoic acid antigens in the bacterial cell wall.

b. Kaufmann- White Scheme- is a system that classifies the

genus Salmonella into serotypes, based on surface antigens.

Diagnostic Testing
a. Schultz-Charlton- A skin test for scarlet fever that uses antitoxin to
the erythrogenic toxin of Streptococcus pyogenes subcutaneously: a
positive reaction is blanching of the rash in the area around the
injection site.

b. Widal test- is one method that may be used to help make

a presumptive diagnosis of enteric fever, also known as typhoid
fever.

c. Weil- Felix test- is an agglutination test for the diagnosis of rickettsial

infections.

Susceptibility Testing
For tuberculosis
a. Mantoux- also known as the Mantoux screening test, tuberculin sensitivity
test, Pirquet test, or PPD test for purified protein derivative) is a tool
for screening for tuberculosis (TB) and for tuberculosis diagnosis.

For diphtheria
a. Schick test- is a test used to determine whether or not a person is
susceptible to diphtheria. It was named after its inventor, Béla
Schick (1877–1967), a Hungarian-born American pediatrician.
The test is a simple procedure. A small amount (0.1 ml) of diluted (1/50 MLD)
diphtheria toxin is injected intradermal into one arm of the person and a heat
inactivated toxin on the other as a control. If a person does not have
enough antibodies to fight it off, the skin around the injection will become red and
swollen, indicating a positive result. This swelling disappears after a few days. If
the person has an immunity, then little or no swelling and redness will occur,
indicating a negative result.
Results can be interpreted as:

1. Positive: when the test results in a wheel of 5–10 mm diameter, reaching

its peak in 4–7 days. The control arm shows no reaction. This indicates
that the subject lacks antibodies against the toxin and hence is
susceptible to the disease.
2. Pseudo-positive: when there is only a red colored inflammation (erythema)
and it disappears within 4 days. This happens on both the arms since the
subject is immune but hypersensitive to the toxin.
3. Negative reaction: Indicates that the person is immune
4. Combined reaction: Initial picture is like that of the pseudo-reaction but the
erythema fades off after 4 days only in the control arm. It progresses on
the test arm to a typical positive. The subject is interpreted to be both
susceptible and hypersensitive.
The test was created when immunizing agents were scarce and not very safe,
however as newer and safer toxoids were made available there was no more
requirement for susceptibility tests.

For anthrax- Ascoli Test

For scarlet fever- Dick’s test
For Streptococcus pneumoniae infection- Francis test
For Glander’s disease- Mallein test
Animal Inoculation

The act or an instance of inoculating, especially the introduction of an anti

genic substance or vaccine into the body toproduce immunity to a specific diseas
e.
Animal inoculation test is used in the following:
a. Ocular test of Anton
a test used in the identification of Listeria monocytogenes; instillation of a
culture into the conjunctival sac of a rabbit orguinea pig causes severe keratocon
junctivitis within 24 hours.
b. To differentiate Streptococcus pneumoniae from viridanas streptococci.
c. Used in the diagnosis of arboviral infections.
d. Used in the diagnosis of chaga’s disease, caused by Trypanosoma cruzi.
Molecular Techniques

In the Molecular era of classification, Carl Woese, who is regarded as the forerunner of
the molecular phylogeny revolution, argued that the bacteria, archaea, and eukaryotes
represent separate lines of descent that diverged early on from an ancestral colony of
organisms. However, a few biologists argue that the Archaea and Eukaryota arose from
a group of bacteria. In any case, it is thought that viruses and archaea began
relationships approximately two billion years ago, and that co-evolution may have been
occurring between members of these groups. It is possible that the last common
ancestor of the bacteria and archaea was a thermophile, which raises the possibility that
lower temperatures are “extreme environments” in archaeal terms, and organisms that
live in cooler environments appeared only later. Since the Archaea and Bacteria are no
more related to each other than they are to eukaryotes, the term prokaryote’s only
surviving meaning is “not a eukaryote”, limiting its value.

With improved methodologies it became clear that the methanogenic bacteria were
profoundly different and were erroneously believed to be relics of ancient bacteria. Thus,
though Woese identified three primary lines of descent the Archaebacteria, the
Eubacteria and the Urkaryotes, the latter now represented by the nucleocytoplasmic
component of the Eukaryotes. these lineages were formalised into the rank Domain
(regio in Latin) which divided Life into 3 domains: the Eukaryota, the Archaea and the
Bacteria. This scheme is still followed today.

In 1987 Carl Woese divided the Eubacteria into 11 divisions based on 16S ribosomal
RNA (SSU) sequences, which with several additions are still used today.

Accurate diagnosis of bacterial infections decreases the spread of the disease and
facilitates appropriate patient management. In addition, efficacious disease treatment
reduces side effects and slows the generation of antibiotic resistance. Traditionally,
culture methods facilitate the diagnosis of most bacterial infections; however, some
bacteria are difficult to isolate, grow slowly in the culture due to stringent growth
requirements, or may not grow because of prior empirical treatment of patients with
antimicrobial agents.1 In these cases, the sensitivity of culture is reduced and the time
from specimen receipt to final report (ie, the turnaround time) is shortened. Hence,
alternative methods capable of overcoming these limitations must be developed. Over
the last 2 decades, bacterial nucleic acid sequence data (ie, genomic and ribosomal)
was obtained and included in numerous databases. This information enabled the
development of several new nucleic acid tests (NATs) for the diagnosis of bacterial
infections. Briefly, NATs use nucleic acid primers or probes that are complementary to
the nucleic acid of the target sequence of the bacteria. These primers or probes bind to
the bacteria’s target sequence, forming a hybrid molecule. Detection of this hybrid can
be accomplished either by direct detection of the hybrid using a reporter molecule (ie,
signal amplification) or enzymatic amplification of the hybrid followed by detection of the
amplified product (ie, target amplification). Development of these assays, therefore,
requires knowledge of the nucleic acid sequences of various isolates of the bacteria of
interest. Specifically, false-positive reactions can result between genetically related
species. Conversely, strains that have nucleic acid changes or variations within their
target sequences can fail to hybridize with the primer or probe, resulting in a false-
negative reaction. Even with these limitations, NAT assays that are more sensitive and
rapid than culture have been developed.
Staining Techniques

In their natural state, most of the cells and microorganisms that we observe under the
microscope lack color and contrast. This makes it difficult, if not impossible, to detect
important cellular structures and their distinguishing characteristics without artificially
treating specimens. We have already alluded to certain techniques involving stains and
fluorescent dyes, and in this section we will discuss specific techniques for sample
preparation in greater detail. Indeed, numerous methods have been developed to identify
specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue
samples, under the microscope. Here, we will focus on the most clinically relevant
techniques.

In clinical settings, light microscopes are the most commonly used microscopes. There
are two basic types of preparation used to view specimens with a light microscope: wet
mounts and fixed specimens.

The simplest type of preparation is the wet mount, in which the specimen is placed on
the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a
liquid form and can be deposited on the slide using a dropper. Solid specimens, such as
a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the
wet mount. Sometimes the liquid used is simply water, but often stains are added to
enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on
top and the specimen is ready for examination under the microscope.

The second method of preparing specimens for light microscopy is fixation. The “fixing”
of a sample refers to the process of attaching cells to a slide. Fixation is often achieved
either by heating (heat fixing) or chemically treating the specimen. In addition to
attaching the specimen to the slide, fixation also kills microorganisms in the specimen,
stopping their movement and metabolism while preserving the integrity of their cellular
components for observation.

In addition to fixation, staining is almost always applied to color certain features of a

specimen before examining it under a light microscope. Stains, or dyes, contain salts
made up of a positive ion and a negative ion. Depending on the type of dye, the positive
or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is
called the counterion. If the chromophore is the positively charged ion, the stain is
classified as a basic dye; if the negative ion is the chromophore, the stain is considered
an acidic dye.

Dyes are selected for staining based on the chemical properties of the dye and the
specimen being observed, which determine how the dye will interact with the specimen.
In most cases, it is preferable to use a positive stain, a dye that will be absorbed by the
cells or organisms being observed, adding color to objects of interest to make them
stand out against the background. However, there are scenarios in which it is
advantageous to use a negative stain, which is absorbed by the background but not by
the cells or organisms in the specimen. Negative staining produces an outline or
silhouette of the organisms against a colorful background (Figure 2).
Figure 2. (a) These Bacillus anthracis cells have absorbed crystal violet, a basic positive stain. (b)
This specimen of Spinoloricus, a microscopic marine organism, has been stained with rose
bengal, a positive acidic stain. (c) These B. megaterium appear to be white because they have
not absorbed the negative red stain applied to the slide.

Because cells typically have negatively charged cell walls, the positive chromophores in
basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly
used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue,
and safranin typically serve as positive stains. On the other hand, the negatively charged
chromophores in acidic dyes are repelled by negatively charged cell walls, making them
negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose
bengal.

Some staining techniques involve the application of only one dye to the sample; others
require more than one dye. In simple staining, a single dye is used to emphasize
particular structures in the specimen. A simple stain will generally make all of the
organisms in a sample appear to be the same color, even if the sample contains more
than one type of organism. In contrast, differential staining distinguishes organisms
based on their interactions with multiple stains. In other words, two organisms in a
differentially stained sample may appear to be different colors. Differential staining
techniques commonly used in clinical settings include Gram staining, acid-fast staining,
endospore staining, flagella staining, and capsule staining.

Gram Stain
The Gram stain procedure is a differential staining procedure that involves multiple
steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an
effective method to distinguish between bacteria with different types of cell walls, and
even today it remains one of the most frequently used staining techniques.
First, crystal violet, a primary stain, is applied to a heat-fixed smear, giving all of the cells
a purple color.
Next, Gram’s iodine, a mordant, is added. A mordant is a substance used to set or
stabilize stains or dyes; in this case, Gram’s iodine acts like a trapping agent that
complexes with the crystal violet, making the crystal violet–iodine complex clump and
stay contained in thick layers of peptidoglycan in the cell walls.
Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol solution.
Cells that have thick peptidoglycan layers in their cell walls are much less affected by the
decolorizing agent; they generally retain the crystal violet dye and remain purple.
However, the decolorizing agent more easily washes the dye out of cells with thinner
peptidoglycan layers, making them again colorless.
Finally, a secondary counterstain, usually safranin, is added. This stains the decolorized
cells pink and is less noticeable in the cells that still contain the crystal violet dye.
Gram-staining is a differential staining technique that uses a primary stain and a
secondary counterstain to distinguish between gram-positive and gram-negative
bacteria.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the
red, safranin-dyed cells are gram-negative. However, there are several important
considerations in interpreting the results of a Gram stain. First, older bacterial cells may
have damage to their cell walls that causes them to appear gram-negative even if the
species is gram-positive. Thus, it is best to use fresh bacterial cultures for Gram staining.
Second, errors such as leaving on decolorizer too long can affect the results. In some
cases, most cells will appear gram-positive while a few appear gram-negative. This
suggests damage to the individual cells or that decolorizer was left on for too long; the
cells should still be classified as gram-positive if they are all the same species rather
than a mixed culture.
Besides their differing interactions with dyes and decolorizing agents, the chemical
differences between gram-positive and gram-negative cells have other implications with
clinical relevance. For example, Gram staining can help clinicians classify bacterial
pathogens in a sample into categories associated with specific properties. Gram-
negative bacteria tend to be more resistant to certain antibiotics than gram-positive
bacteria.
Acid-Fast Stains
Acid-fast staining is another commonly used, differential staining technique that can be
an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-
positive cells: those that have waxy mycolic acids in their cell walls, and those that do
not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and
the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast
cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is
applied. A secondary counterstain, methylene blue, is then applied, which renders non–
acid-fast cells blue.
The fundamental difference between the two carbolfuchsin-based methods is whether
heat is used during the primary staining process. The Ziehl-Neelsen method uses heat
to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not
use heat. Both techniques are important diagnostic tools because a number of specific
diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample,
their red or pink color can be seen clearly against the blue background of the
surrounding tissue cells.
Capsule Staining
Certain bacteria and yeasts have a protective outer structure called a capsule. Since the
presence of a capsule is directly related to a microbe’s virulence (its ability to cause
disease), the ability to determine whether cells in a sample have capsules is an
important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a negative
staining technique (staining around the cells) is typically used for capsule staining. The
dye stains the background but does not penetrate the capsules, which appear like halos
around the borders of the cell. The specimen does not need to be heat-fixed prior to
negative staining.
One common negative staining technique for identifying encapsulated yeast and
bacteria is to add a few drops of India ink or nigrosinto a specimen. Other capsular
stains can also be used to negatively stain encapsulated cells. Alternatively, positive and
negative staining techniques can be combined to visualize capsules: The positive stain
colors the body of the cell, and the negative stain colors the background but not the
capsule, leaving halo around each cell.
Endospore Staining
Endospores are structures produced within certain bacterial cells that allow them to
survive harsh conditions. Gram staining alone cannot be used to visualize endospores,
which appear clear when Gram-stained cells are viewed. Endospore staining uses two
stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton
method (the most commonly used endospore-staining technique) uses heat to push the
primary stain (malachite green) into the endospore. Washing with water decolorizes the
cell, but the endospore retains the green stain. The cell is then counterstained pink
with safranin. The resulting image reveals the shape and location of endospores, if they
are present. The green endospores will appear either within the pink vegetative cells or
as separate from the pink cells altogether. If no endospores are present, then only the
pink vegetative cells will be visible.
Flagella Staining
Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some
bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be
seen under a light microscope without a specialized flagella staining technique. Flagella
staining thickens the flagella by first applying mordant (generally tannic acid, but
sometimes potassium alum), which coats the flagella; then the specimen is stained
with pararosaniline (most commonly) or basic fuchsin.
Though flagella staining is uncommon in clinical settings, the technique is commonly
used by microbiologists, since the location and number of flagella can be useful in
classifying and identifying bacteria in a sample. When using this technique, it is
important to handle the specimen with great care; flagella are delicate structures that
can easily be damaged or pulled off, compromising attempts to accurately locate and
count the number of flagella.
Reference
https://courses.lumenlearning.com/boundless-microbiology/chapter/microbial-culture-
methods/
https://courses.lumenlearning.com/boundless-microbiology/chapter/classification-of-
microorganisms/
https://watermark.silverchair.com/labmed39-0430.pdf
https://microbiologyinfo.com/techniques-of-virus-cultivation/
https://courses.lumenlearning.com/microbiology/chapter/staining-microscopic-
specimens/