This document discusses various microbiological techniques used to study microorganisms, including: 1. Microscopic, cultural, physiological, immunological, and molecular methods. Specific techniques mentioned are Gram staining, growth media selection, enzyme activity assays, immunoassays, DNA fingerprinting, gene probes, microarrays, PCR, and metagenomics. 2. 16S rRNA gene sequencing is described as the most widely used molecular technique for bacterial identification and phylogenetic analysis due to the conserved nature of the 16S rRNA gene. 3. Metagenomics provides information on the collective genomes of microorganisms in an environmental sample to study microbial diversity and ecology.

1. Akash Mahadev Iyer
2nd semester
M.Sc Biochemistry
University of Kerala

2. 1. Microscopic methods
2. Cultural methods
3. Physiological methods
4. Immunological methods
5. Molecular methods (Nucleic acid
based methods)

4. Bright Field
Dark Field
Phase contrast

5. Gram’s Staining
Flagella Staining
Endospore Staining Negative Staining

6. MFT
Anaerobic Cultivation

9. Media Organism
XLD agar Salmonella
S1 medium (Sucrose, SLS,
casamino acids, Trimethoprim)
Fluorescesnt
Pseudomonads
Agar medium containing K+ buffers,
Mg2+ salts, Trace elements, NaNO2,
Bromothymol blue
Nitrosomonas and
Nitrobacter
BG11 medium BGA
Bold’s Basa medium
(+2000-3000 lux light)
Green and yellow-green
Algae
PDA, MEA
(+Rose Bengal)
Fungi

10. • This medium supports growth of photoautotrophic blue green algae .
• They require light as source of energy. Synthetic nitrogen and carbon
sources and other inorganic salts comprise this medium.
• Exposure to light intensity of 2,000 to 3,000 lux is optimal for cultivation
of blue green algae. Neon light source is found to be sufficient to provide
this illumination. For maintenance of blue green algae exposure for
period of 24 hours a day is optimal .Often the flasks kept for incubation
may be covered with grease proof paper. They grow optimally at room
temperature between range of 20-25°C.

11.  Provides information about what activities are carried out
in microbial communities- Contribution of microbe in the
environment
 Detection and characterization of unknown compounds-
MS
 Distinguish metals- , eg; Measures oxidation of S0
to sulphate.
 Estimates sulphate as a precipitate with Barium chloride
DO- Based on precipitation of dissolved oxygen
using
 Manganous sulphate and a KOH-KI mixture
 The oxygen precipitate, MnO2 reacts with sulfuric acid to form
manganic sulphate which inturn reacts with KI to liberate iodine
 No. of moles of Iodine No. of moles of oxygen present

14.  Cell mass- Turbidimetric method
 Measure protein- Folin lowry, BCA, Bradford assay

16. Enzymes-Dehydrogenase (TTC), Esterase (FDA)

18. Immunological methods

19. Immunoaffinity Chromatography
•Immobilisation of antibody onto
a matrix, normally beads, which
are then placed into a
chromatography column.
•The columns are made by
reacting highly purified antibody
(monoclonal or polyclonal) with
the chromatography beads to
form the affinity matrix.

20. Immunofluorescence

21. Phage Typing
 Determining which phages a
bacterium is susceptible to.
 The tested strain was grown
over entire plate; known phages
are placed in different squares;
plaques (areas of lysis) appear
dark indicating sensitivity to a
specific phage

22. Flow cytometry
FACS

23.  DNA base composition- Guanine + cytosine moles% (GC)
 DNA fingerprinting- Electrophoresis of restriction enzyme digests
 rRNA sequencing
 Gene Probing- ssDNA fragments to identify particular nucleic acid
sequence
 Hybridization
 Microarray
 Polymerase chain reaction (PCR)
 Metagenomics

24. Fingerprinting-

25. Gene probe based
detection of DNA-
Colorimetric,
Fluorescence,
Chemiluminesence

26. Microarray
 A microarray is a laboratory tool
used to detect the expression of
thousands of genes at the same
time.
 DNA microarrays are microscope
slides that are printed with
thousands of tiny spots in
defined positions, with each spot
containing a known DNA
sequence or gene.
 Often, these slides are referred to
as gene chips or DNA chips.
 The DNA molecules attached to
each slide act as probes to
detect gene expression.

28. FISH Analysis of Sewage Sludge

29. PCR

30. 16s rRNA sequence analysis
 16S rRNA most conserved (least
variable) in all cells.
 Portions of the sequence from
distantly related organisms are
remarkably similar.
 Sequences from distantly related
organisms can be precisely
aligned, making the true
differences easy to measure.
 Genes that encode the rRNA
(rDNA) have been used
extensively to determine
taxonomy, phylogeny
(evolutionary relationships), and
to estimate rates of species
divergence among bacteria.
Thus, the comparison of 16s rRNA
sequence can show evolutionary
relatedness among
microorganisms.

31. RIBOSOMAL RNA •
 To infer relationships that span the diversity of known life, it is necessary
to look at genes conserved through the billions of years of evolutionary
divergence.
 Examples of genes in this category are those that define the ribosomal
RNAs (rRNAs).
 In Bacteria, Archaea, Mitochondria, and Chloroplasts, the small ribosomal
subunit contains the 16S rRNA (where the S in 16S represents Svedberg
units).
 The large ribosomal subunit contains two rRNA species (the 5S and 23S
 rRNAs).
 Most prokaryotes have three rRNAs, called the 5S, 16S and 23S rRNA.
Bacterial 16S, 23S, and 5S rRNA genes are typically organized as a co-
transcribed operon.
 There may be one or more copies of the operon dispersed in the genome
(for example, E coli has seven).
 The Archaea contains either a single rDNA operon or multiple copies of the
operon rRNA targets were studied originally, most researchers now target
the corresponding ribosomal DNA (rDNA) because DNA is more stable and
easier to analyse

32.  Conventional microbiology techniques -to identify most of the
bacteria, fungi and other pathogens- about 8 to 20 hours for an
accurate result.
 The presence of hyper variable regions in the 16S rRNA gene
provides a species specific signature sequence which is useful for
bacterial identification process.

33. Metagenomics is the study of the metagenome—the collective genome
of microorganisms from an environmental sample—to provide
information on the microbial diversity and ecology of a specific
environment.
 DNA is cloned from microbial community and sequenced
 Detects as many genes as possible
 Yields picture of gene pool in environment
 Powerful tool for assessing the phylogenetic and metabolic diversity
of an environment