Microbiology

INTRODUCTION TO
MICROBIOLOGY
By
A. Nandakumar, M.tech.,
AP/BIOTECH
KIT-CBE.

Microbiology
 It is the study of living organisms of microscopic size, which
includesbacteria, fungi,algae,protozoa and viruses.
 It is majorly concerned with classification, their forms, structure,
reproduction, physiology, metabolism and their association
withnature.
 Microorganisms are both beneficial and causes harm to other
living organisms.
 They are unicellularand contains singlecells.

Historical Outbreaks….
S. No. Period Scientists/ Community Contribution
1. 1546 Fracastoro
Suggeststhatinvisible organismscause
disease
2. 1590-1608 Jansen Developsfirstuseful compoundmicroscope
3. 1665 Hooke Micrographia
4. 1676 Leeuwenhoek Discovers“animalcules”(Light Microscope)
5. 1688 Redi Spontaneousgenerationofmaggots
6. 1765–1776 Spallanzani Attacksspontaneousgeneration
7. 1786 Müller First classificationofbacteria
8. 1798 Jenner Cowpoxvaccinationforsmallpox
9. 1838–1839 SchwannandSchleiden TheCell Theory
10. 1835–1844 Bassi
Discoversthatsilkwormdiseaseiscaused
byafungus andproposesthatmany
diseases aremicrobial in origin

S.
No.
Period
Scientists/
Community
Contribution
11. 1847–1850 Semmelweis
Showsthatchildbedfever istransmittedbyphysiciansand
introducestheuseof antisepticstoprevent thedisease
12. 1849 Snow Studiesthe epidemiology ofcholeraepidemic in London
13. 1857 Pasteur Lacticacidfermentation
14. 1858 Virchow All cells arecomefromcells
15. 1861 Pasteur Disprovesspontaneousgeneration
16. 1867 Lister Antisepticsurgery
17. 1869 Miescher Nucleic acids
18. 1876-1877 Koch Anthraxis causedbyBacillusanthracis
19. 1880 Laveran Plasmodium (causeofmalaria)
20.
1881
Koch Culturesbacteriaongelatin
21. Pasteur Anthraxvaccine

22. 1882 Koch Mycobacteriumtuberculosis
23.
1884
Koch KochPostulates
24. Metchnikoff Phagocytosis
25. Autoclavedeveloped
26. Gram Gram’sstain
27.
1885
Pasteur Rabiesvaccine
28. Escherich E.coli
29. 1886 Fraenkel Streptococcuspneumoniae
30. 1887 RichardPetri Petridish (plate)
31. 1887-1890 Winogradsky Sulfurandnitrifyingbacteria
32. 1889 Beijerinck Isolatesrootnodulebacteria
33. 1890 VonBehring antitoxinsfordiphtheriaandtetanus

34. 1892 Ivanowsky
Providesevidence forvirus causationof
tobaccomosaicdisease
35. 1894 KitasatoandYersin Yersiniapestis
36. 1896 VanErmengem Clostridiumbotulinum
37.
1897
Buchner extractofyeast
38. Ross Malariaparasiteiscarriedbythe mosquito
39. 1900 Reed Yellow feveris transmittedbythe mosquito
40. 1905 SchaudinnandHoffmann Treponemapallidumcausessyphilis
41. 1906 Wassermann Fixationtestforsyphilis
42. 1909 Ricketts Rickettsiarickettsii
43. 1910 Ehrlich Chemotherapeuticagent forsyphilis
44. 1915-1917 D’Herelle andTwort Bacterialviruses
45. 1921 Fleming Lysozyme

46. 1923 - First editionofBergey’sManual
47. 1928 Griffith Bacterialtransformation
48. 1929 AlexanderFleming Penicillin
49. 1931 VanNiel
Photosyntheticbacteriausereducedcompounds
aselectron donorswithoutproducingoxygen
50. 1933 Ruska First transmissionelectron microscope
51.
1935
Stanley Crystallizesthe tobaccomosaicvirus
52. Domagk Sulfadrugs
53. 1937 Chatton
Divides living organismsintoprokaryotesand
eukaryotes
54. 1941 Beadle andTatum One-gene-one-enzymehypothesis
55.
1944
Avery DNAcarries informationduring transformation
56. Waksman Streptomycin

57. 1946 Lederberg andTatum Bacterialconjugation
58. 1949
Enders,Wellerand
Robbins
Growpoliovirus in humantissuecultures
59. 1950 Lwoff Lysogenicbacteriophages
60.
1952
Hershey andChase Bacteriophagesinject DNAintohostcells
61. ZinderandLederberg Generalizedtransduction
62.
1953
- Phasecontrast microscope
63. WatsonandCrick Double-helixStructureofDNA
64.
1955
JacobandWollman Ffactoris a plasmid
65. Jerne andBurnet Theclonalselection theory
66. 1959 Yalow Radioimmunoassaytechnique
67. 1961 JacobandMonod Operonmodel ofgene regulation
68. 1961-1966 Nirenberg,Khorana elucidate thegenetic code

69. 1962 - First quinoloneantimicrobial(nalidixic acid)
70.
1970
ArberandSmith Restrictionendonucleases
71. Temin andBaltimore Reversetranscriptasein retroviruses
72.
1973
Ames Bacterialassayforthedetectionof mutagens
73.
Cohen,Boyer,Chang,and
Helling
Plasmidvectorstoclone genes in bacteria
74. 1975 - Lymedisease
75. 1977 WoeseandFox
Recognitionofarchaeaasa distinctmicrobial
group
76. 1979 - Smallpoxofficiallyeliminated
77. 1980 - Scanningtunneling microscope
78. 1982 - RecombinanthepatitisBvaccine
79.
1983-1984
Gallo andMontagnier Human immunodeficiencyvirus
80. Mullis Polymerasechainreaction

81. 1986 -
First vaccine (hepatitisBvaccine) producedby
genetic engineering approvedforhumanuse
82.
1995
U.S. Chicken poxvaccine
83. - Haemophilusinfluenzaegenome sequenced
84. 1996 -
Methanococcusjannaschiigenome sequenced
Yeastgenome sequenced
85.
1997
-
Thiomargaritanamibiensis largestknown
bacteria
86. - E.coli genomesequenced
87.
2000
-
Vibriocholeraehastwoseparate
chromosomes
88. Wyeth PrevnarVaccineforpneumonia
89.
2001
Asia Footandmouthdisease
90. JoergC. Tiller Hexyl-PVPforkilling microbes

91. 2001 Ekata(Congo Border) Ebolavirus outbreak
92.
2002
NIH Staphvaccine
93. - VancomycinresistantStaphylococcusaureus
94. 2003 - SARS
95. 2004 Hong Kong Avaininfluenza(H5N1 Avain flu)
96. 2005 - Nipahvirus
97.
2007
China Swinevirus
98. - Ebola
99. 2014 Caribbeanislands Chikungunyavirus
100. 2016 America Zikavirus
101. 2018 French drug makerSanofi Anti-denguevaccine (Dengvaxia)

Antony van Leeuwenhoek…
 The “Father ofMicrobiology”(1632-1723).
 Best knownfor his contribution to the improvement of the microscope and for
his contributions towards the establishment ofmicrobiology.
 He firstobserveda singlecelledorganisms named as “animalcules”.
 His observations are with bacteria, muscle fibers, microscopic nematodes,
rotifers,spermatozoa, blood cells and small blood vessels.
 He discovered over five hundred optical lenses and over four hundred different
types ofmicroscopes, nineis stillinexistence.
 His microscopes are made up of silver or copper metal frames and able to
magnifyup to 270 times. (Twoinches longand one inch)

In 1673, his association with English Royal Society and published his
earliest contribution in its journal, “PhilosophicalTransactions”.
In 1680, he appointed asa member of the Royal society.
In 1674, he discovered infusoria (dated zoological category), the minute
organisms in freshwater pond water, including description of the green
alga Spirogyra.
In 1676, he discovered bacteria.
In 1677, he discovered spermatozoa in humans and
other mammals and in molluscs, fish, amphibians, and birds. He then
came to the novel conclusion that fertilization occurred when
spermatozoa penetrated the egg.

 In1682, van Leeuwenhoek discovered the banded patternof muscular fibers.
 In 1683, he described his microscopic examination of the microorganisms
found inthe plaque on hisown teeth, and on twowomen, and twooldmen.
 In 1702, van Leeuwenhoek described many protists, including the
cilite, Vorticella. Van Leeuwenhoek also described the mouthparts of insects,
parthenogenesis in aphids, the minute globular particles in yeast, and gave the
first accurate, detailed description of red blood cells. His study of mussels,
weevils, and fleas helped refute the accepted doctrine of spontaneous
generation.

Van Leeuwenhoek has been attributed as the first person to use
a histological stain to color specimens observed under the
microscope using saffron.
Like Robert Boyle and Nicolaas Hartsoeker, van Leeuwenhoek was
interested in dried cochineal, trying to find out if the dye came from
a berry or aninsect.
Van Leeuwenhoek's religion was "Dutch Reformed" Calvinist. He
often referred with reverence to the wonders God designed in making
creatures great and small, and believed that his discoveries were
merely further proof of the wonder of creation.
At the end, he hascontributed 560 letters to Royal Society

Spontaneous Generation…
Aristotle (384-322 BC) – the theory of spontaneous generation.
That life can arise from non-living matter, if it contains pneuma (Vital
heat).
During 17th century van Helmont, mice arise from rags and wheat
kernels left in anopen container for 3 weeks.
But in reality, it provides shelter and nourishment to mice.
Francesco Redi in 1668, performed an experiment to disprove the
idea that maggots (larvae of flies) spontaneous generate on meat
left out in the open air.

Spontaneous Generation…
In 1745, John Needham, in his experiment, he briefly boiled broth
infused with plant or animal matter, hoping to kill all preexisting
microbes.
He then sealed the flasks. After a few days, Needham observed that
the broth had become cloudy and a single drop contained numerous
microscopic creatures.
He argued that the new microbes must have arisen spontaneously.
In reality, however, he likely did not boil the broth enough to kill all
preexisting microbes.

Lazzaro Spallanzani…
Italian biologist and physiologist (1729-1799).
His work about experimental study of bodily functions, animal
reproduction andanimalecholocation.
He refuted the spontaneous generation (1767) with the help of
biogenesis, which leads the way for Louis Pasteur.
In a series of experiments he showed that gravy, when boiled, did not
produce these forms if placed in phials that were immediately sealed
by fusing the glass.
As a result of this work, he concluded that the objects in pond water
andother preparations were livingorganisms introduced from the air.
Studied various forms of Leeuwenhoek microscopic life and
confirmed that they are livingorganisms.

In 1768, regenerationstudies wascarried out.
Conclusions: Lower animals have greater generative power than higher
animals.
Young individuals have greater capacity for regeneration than adults of
same species; except its superficial parts not the internal organs can
regenerate.
Transplantation experiments showed great experimental skill and
included the successful transplant of the head of one snail onto the body
of another.
In 1773, he investigated the circulation of the blood through the lungs
and other organs and did an important series of experiments
on digestion, in which he obtained evidence that digestive juice
contains special chemicals that are suited to particular foods.

Spermatazoa wereparasites withinthesemen.
Preformation theory was accepted by him, according to this
theory, the germs of all living things was created b y God and
encapsulatedwithinfirst femaleof each species.
Expansionof egg is thestimulusgivenby thesperm cells.
Egg and sperm contact is important for the development of
new animals.
Performed some of the first successful artificial
inseminationexperimentson lower animalsand on a dog.

Louis Pasteur…
French chemist andMicrobiologist (1822-1895).
One of the Foundersof Medical Microbiology.
He studied the molecular asymmetry, microorganisms involved in
fermentation and disease, Pasteurization, also saved beer, wine and
silkindustries in France, vaccinesfor Rabies and anthrax.
In 1854, alcoholicfermentation (Lactic acid and Butyric acid).
In 1857, he proved that the living organisms responsible for
fermentation process(Specific organisms for specific fermentation).
Germtheory of fermentation.
Sterilizing and cleaning in medical practices and antiseptic methods in
surgery.

Louis Pasteur…
Specific organisms involved in fermentation was identified in butyric
acidfermentation.
It led to discovery that the fermentation process could be arrested by
passing air through the fermenting fluid, a process known today as the
Pasteur effect.
He concluded that life-form that could function only in the absence of
oxygen.
Aerobicandanaerobic organisms.
Putrefaction due to specific germs under
anaerobic conditions.

Louis Pasteur…
In 1863, Pasteur studied wine contamination and showed it was
causedby microbes.
To prevent contamination, Pasteur used a simple procedure: he
heated the wine to 50–60 °C (120–140 °F), a process now known
universally aspasteurization.
But this is not used for wine, used for food products andbeverages.
For brewing industries, he devised a method for the manufacturing
process that prevents deterioration.
He attacks the spontaneous generation by using “Swan-flask
culture”.
Preservation of healthy silkworm eggs and prevented their
contamination by the disease-causingorganisms.

Louis Pasteur…
1870’s – Vaccineproduction.
Pasteur developed the overall principle of vaccination and contributed
to the foundation of immunology.
1879 – chicken cholera vaccination, which is a live attenuated
cultures.
Chicken cholera cultures lost their pathogenicity and retained
“Attenuated” pathogenic characteristics over course of many
generations.
He inoculated chickens with the attenuated form and demonstrated
that the chickenswere resistant to the fully virulent strain.
Rectified problem of immunization.

Louis Pasteur…
In 1879 – Bacillusanthraxresponsible for the infection.
Vaccination for anthrax was discovered with two stages (One with
lower virulent strains andanother with more virulent strains).
In 1882 – first vaccination against rabies (inactivated vaccines), the
era of preventivemedicine.
In 1888, Pasteur institute in Paris was originated.

Robert Hooke…
1635-1703– Englishphysicist.
First person to report seeing microbes under the microscope.
Working with a crude compound microscope he saw the cellular
structure of plantsaround1665.
His reputation in history largely rests on his Book Micrographia,
publishedin1665.
Hooke devises the compound microscope and illumination system
oneofthebest such microscopes inhis time.
He also saw fungi which he drew. However, because his lens were
of poorqualityhe wasapparently unable to“see” bacteria.
He discovered plant cells, more precisely they were cell walls of
corktissue. Infactit wasHookewhocoinedthe term “Cell”.

Robert Hooke…
Hooke was asked by society to confirm Leeuwenhoek’s findings
whichhe successfully did.
He also studied fossil molluscans and concluded that there were
organismswhichlived onearth earlier but are nowextinct.
Thus he should be regarded as a pioneering paleontologist who
actually understood an important aspect of biology that the fossils
are evidence of evolution. Hooke even studied fossils under his
compoundmicroscope.

Robert Koch…
Founder of bacteriology andmicrobiology (1843-1910).
Anthrax disease cycle – 1876.
Bacteriafor tuberculosis – 1882. (Awarded noble prize in 1905)
Cholera – 1883.
Anthrax :
He cultivated the anthrax organisms in suitable media on
microscope slides, demonstrated their growth into long filaments,
and discovered the formation within them of oval, translucent
bodies—dormant spores.
The dried spores could remain viable for years, even under
exposed conditions.
Even dormant spores could cause disease to the living organisms.

Robert Koch…
The pure culture techniques were emerged and also the specific
disease was caused by specific organism was confirmed later.
In 1877, published on the investigation, preservation, and
photographing of bacteria.
He described his method of preparing thin layers of bacteria on glass
slides and fixingthem by gentle heat.
Koch also invented the apparatus and the procedure for the very
useful hanging-drop technique, whereby microorganisms could be
cultured in a drop of nutrient solution on the underside of a glass
slide.

Robert Koch…
In 1878, he summarized experiments on the etiology of wound
infection.
By inoculating animals with material from various sources, he
produced six types of infection, each caused by a specific
microorganism.
He then transferred these infections by inoculation through several
kindsof animals,reproducing the original sixtypes.
In that study, he observed differences in pathogenicity for different
species of hosts and demonstrated that the animal body is an excellent
apparatus for the cultivation of bacteria.

Robert Koch…
Koch determined guidelines to prove that a disease is caused by a
specific organism. These four basic criteria, called Koch’s postulates,
are:

Robert Koch…
Koch concentrated his efforts on the study of tuberculosis, with the
aim of isolating its cause.
Koch discovered the tubercle bacillus and established its presence in
the tissues of animals and humans suffering from the disease, which
he believed to be the causeof allforms of tuberculosis.
In 1890, he used as the active agent a sterile liquid produced
from cultures of the bacillus.
However, the liquid, which he named tuberculin, proved
disappointing, and sometimes dangerous, as a curative agent.
A particular comma-shaped bacterium (vibrio) as the cause of cholera,
the epidemic ended before he was able to confirm his hypothesis.
In India, where cholera is endemic, he completed his task, identifying
both the organism responsible for the disease and its transmission via
drinking water, food, andclothing.

Edward Jenner…
Father of immunology (1749-1823).
Pioneer of smallpox vaccine.
During 18th century, the only means of combating smallpox was a
primitive form of vaccination called variolation—intentionally
infecting a healthy person with the “matter” taken from a patient sick
with a mild attackof the disease.
But it was already originated in INDIA and CHINA, based on two
distinct concepts: first, that one attack of smallpox effectively
protected against any subsequent attack and, second, that a person
deliberately infected with a mild case of the disease would safely
acquire suchprotection.

Edward Jenner…
Jenner had been impressed by the fact that a person who had suffered
an attack of cowpox—a relatively harmless disease that could be
contracted from cattle—could not take the smallpox—i.e., could not
become infected whether by accidental or intentional exposure to
smallpox.
Pondering this phenomenon, Jenner concluded that cowpox not only
protected against smallpox but could be transmitted from one person
to another as a deliberate mechanism of protection.
In 1796, he found an young lady with fresh cowpox lesions on her
hand, later he inoculated an eight-year-old boy, who had never had
smallpox.

Edward Jenner…
After 9 days of incubation, that boy became slightly ill.
Then, he inoculated the boy again, this time with smallpox matter.
No disease developed;protection was complete.
In 1980, the World Health Organization declared smallpox an
eradicated disease. This was the result of coordinated public health
efforts by many people, but vaccination was anessentialcomponent.
Known: that smallpox was more dangerous than variolation and
cowpox less dangerous than variolation.
The hypothesis tested: That infection with cowpox would give
immunity to smallpox.
The test: If variolation failed to produce an infection, Phipps was
shown to be immune to smallpox.
The consequence: Immunity to smallpox could be induced much
more safely.

Alexander Fleming…
Bacteriologist (1881-1955), best known for his discovery
of penicillin.
1945, he was awarded noble prizefor physiology ormedicine.
His work on wound infection and lysozyme, an
antibacterial enzyme found in tears and saliva, guaranteed him a place
in the history of bacteriology.
In 1921, lysozyme was discovered.
It came about when he had a cold and a drop of his nasal mucus fell
onto a culture plate of bacteria.
Realizing that his mucus might have an effect on bacterial growth, he
mixed the mucus into the culture and a few weeks later saw signs of
the bacteria’s having been dissolved.
Unfortunately, lysozyme had no effect on the most-pathogenic
bacteria.

In 1928, Fleming noticed that a culture plate of Staphylococcus
aureus, he had been working on had become contaminated by
a fungus.
A mold, later identified as Penicillium notatum (now classified as P.
chrysogenum), had inhibited the growth of the bacteria. He at first
called the substance “mould juice” and then “penicillin,” after the
mold that produced it.
He thought that he had found anenzyme more potent thanlysozyme.
In fact, it was not an enzyme but an antibiotic—one of the first to
be discovered.
Both as a topical antiseptic and as an injectable antibiotic, if it could be
isolated andpurified. Used during WW-II

For the last decade of his life, Fleming was feted universally for his
discovery of penicillin and acted as a world ambassador for medicine
andscience.

Light Microscope…
Resolutionis defined as the ability to distinguish two very small and closely-
spacedobjectsasseparateentities.
Resolutionisbestwhenthedistanceseparatingthetwotinyobjectsissmall.
Degreetowhichdetailinspecimenisretainedinmagnifiedimage.
Resolving power- – Unaided eye – 0.1 mm apart – Microscope - 0.2 µm
apart
Mmicroscope = Moccular X Meyepiece

Electron Microscope…
An electron microscope is a microscope that uses a beam of
accelerated electrons asa source of illumination.
The wavelength of an electron can be up to 100,000 times shorter
than thatof visiblelight photons.
Electron microscopes have a higher resolving power than light
microscopes and canreveal the structure of smaller objects.
It offers unique possibilities to gain insight into 1. Structure 2.
Topology 3. Morphology 4. Composition of materials.
Advantages Of Electron Microscopy
To study objects of >0.2 micrometer.
For analysisof subcellular structure.
For study of intracellularpathogens & viruses.
For cellmetabolism.
For study of minute structure in nature.

Transmission Electron Microscope (TEM)…
The original form of electron microscopy, Transmission electron
microscopy (TEM) involves a high voltage electron beam emitted by an
electron gun, usually fitted with a tungsten filament cathode as the
electronsource.
The electron beam is accelerated by an anode with respect to the
cathode, focused by electrostatic and electromagnetic lenses, and
transmitted through a specimen that is in part transparent to electrons and
inpartscattersthemoutofthebeam.
When it emerges from the specimen, the electron beam carries information
about the structure of the specimen that is magnified by the objective lens
systemofthemicroscope.
The spatial variation in this information (the "image") is recorded by
projecting the magnified electron image onto a fluorescent viewing
screencoatedwitha phosphororscintillatormaterialsuchaszincsulfide.

Transmission Electron Microscope (TEM)…

Scanning Electron Microscope (SEM)…
The Scanning Electron Microscope (SEM) produces images by
detecting low energy secondary electrons which are emitted from
the surface of the specimen due to excitation by the primary electron
beam.
 In the SEM, the electron beam is rastered across the sample, with
detectors building up an image by mapping the detected signals with
beam position.
The TEM resolution is about an order of magnitude greater than the
SEM resolution, however, because the SEM image relies on surface
processes rather than transmission it is able to image bulk samples
and has a much greater depth of view, and so can produce images that
are a good representation of the 3D structure of the sample.

Scanning Electron Microscope (SEM)…

Fluorescence Microscope…
A fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition to,
reflection and absorption to study properties of organic or inorganic
substances.
The "fluorescence microscope" refers to any microscope that uses
fluorescence to generate an image.
When certain compounds are illuminated with high energy light, they
then emit light of a different, lower frequency.
Thiseffect is known as fluorescence.
Often specimens show their own characteristic auto- fluorescence
image, based on their chemical makeup.
Specimens usuallystained with fluorochromes.

Need for Staining …
 Microorganisms can be examined with light microscope
directly.
 But for better visualization – Fixationand Staining required.
 Also to check the morphological characters and for preserving
as specimens.

Fixation
 The internal and external structure of cells and microbes are preserved and fixed in
position.
 Inactivating the enzymes which disrupts the cell morphology for better observation
and staining.
 Therearetwo types.
◦ Heat Fixation
 For prokaryotic cells
 Thinfilm of cells (Usingheat)
 Preservescell morphology, notthestructureswithin thecells.
◦ Chemical Fixation
 Protectfine cellular structureand morphology
 Fixatives penetrate the cells and react with cellular components (proteins
&lipids) make them immobile, inactive and insoluble.
 Ex: Ethanol, Acetic acid, mercuric chloride, formaldehyde and
glutraldehyde.

Dyes & Simple Staining
 Two common groups:
◦ Chromophoregroup:
 Givescolor to cells by ionic, covalent/hydrophobic bonding.
◦ Negative staining:
 Background was stained left the cells without staining (Bright
objects against dark background).
Examples:
Acidic dyes: Eosin, RoseBengal and Acid fuchsine (-vecharged)
Basic dyes: Methylene blue, Basic fuchsine, Crystal Violet, Safranin,
Malachite Green(+vecharged)
 To analyze the size, shapeand arrangement of prokaryotic cells.

Differential Staining
Gram’s Staining:
Based on the cell wall characteristics – Differentiate Gram Positive
and Gram Negative organisms.

Differential Staining
Acid Fast Staining:
Identify Mycobacterium sp. (Contains mycolic acids in the cell wall)
– preventsbinding of dyes to cells.
Ziehl - Neelsen method

Specific Staining
Capsular Staining:
Present onthe cell wall of bacteria and protects them.
Visualized by negative staining.

Specific Staining
Endospore Staining:
Needsharshtreatment to drivedye into a target.
Resistant structure developed by some bacteria (Bacillus &
Clostridium) -Schaeffer–Fulton stain and the Moellerstain.

Specific Staining
Flagella Staining:
It provides taxonomically valuable information about the presence
& distribution of flagella on prokaryotic cells.
Theyare fine, thread-like structure used for locomotion.
To view in microscope, the thickness of the flagella is increased by
coating with mordants like tannic acid and potassium alum & then
with pararosaniline (Leifson method) or basic fuschine (Grey
method).

Microbiology

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