Candida is a yeast and the most common cause of opportunistic mycoses worldwide. It is also a frequent colonizer of human skin and mucous membranes.

1. Sam Higginbottom University of
Agriculture, Technology And Sciences
(SHUATS)
Jacob Institute of Biotechnology and Bio-Engineering
(JIBB)
Department of Industrial Microbiology
Anjali Tripati (16BSMBH012)

2. Genetic diversity analysis of CANDIDA SPECIES
using RAPD MARKERS

3. CANTENT
• Introduction
• Species
• Morphology
• Candida albicans
• Candida tropicalis
• To analyse the morphology of Candida albicans and Candida
tropicalis
- Requirements
- Procedure
- Result
- Fungal staining and result
• To confirm candida isolates using germ tube test
• Sub culturing of the microorganism
• DNA Isolation

4. CONTENT
• Qualitative analysis of DNA
– Agarose gel electrophoresis
• Quantitative analysis of DNA
- Spectroscopy
• Purity of the DNA
• DNA concentration
• RAPD assay
• List of primers used
• DATA analysis
• Result of PCR
• Conclusion
• Disscussion

5. INTRODUCTION
Description and Natural Habitats
• Candida is a yeast and the most common cause of
opportunistic mycoses worldwide.
• It is also a frequent colonizer of human skin and mucous
membranes.
• Candida is a member of normal flora of skin, mouth,
vagina, and stool.
• As well as being a pathogen and a colonizer, it is found in
the environment, particularly on leaves, flowers, water, and
soil.
• While most of the Candida spp. are mitosporic, some have
known teleomorphic state and produce sexual spores.

6. TAXONOMIC CLASSIFICATION
Kingdom: Fungi
Phylum: Ascomycota
Subphylum: Ascomycotina
Class: Ascomycetes
Order: Saccharomycetales
Family: Saccharomycetaceae
Genus: Candida

7. SPECIES
• The genus Candida includes around 154 species. Among
these, six are most frequently isolated in human infections.
• While Candida albicans is the most abundant and
significant species, Candida tropicalis, Candida
glabrata, Candida parapsilosis, Candida krusei, and Candida
lusitaniae are also isolated as causative agents
of Candida infections.
• Importantly, there has been a recent increase in infections
due to non-albicans Candidaspp., such as Candida
glabrata and Candida krusei.

8. Morphology
• The colonies of Candida spp. are cream colored to
yellowish, grow rapidly and mature in 3 days.
• The texture of the colony may be pasty, smooth, glistening
or dry, wrinkled and dull, depending on the species.
• The microscopic features of Candida spp. also show
species-related variations.
• All species produce blastoconidia singly or in small clusters.
Blastoconidia may be round or elongate.
• Most species produce pseudohyphae which may be long,
branched or curved.
• True hyphae and chlamydospores are produced by strains
of some Candida spp.

9. CANDIDA ALBICANS
• Candida albicans is an opportunistic pathogenic yeast that
is a common member of the human gut flora.
• It does not proliferate outside the human body. It is
detected in the gastrointestinal tract and mouth in 40–60%
of healthy adults.
• It is usually a commensal organism, but it can
become pathogenic in immunocompromised individual
under a variety of conditions.
• It is one of the few species of the genus Candida that
causes the human infection candidiasis,which results from
overgrowth of fungus.

10. • C. albicans is commonly used as a model organism for
biology.
• It is generally referred to as a dimorphic fungus since it
grows both as yeast and filamentous cells.
• However, it has several different morphological
phenotypes.
• C. albicans was for a long time considered an obligate
diploid organism without a haploid stage.
• Next to a haploid stage C. albicans can also exist in a
tetraploid stage.
• The diploid genome size is approximately 29 Mb, and up to
70% of the protein coding genes have not yet been
characterized.
• C. albicans is easily cultured in the lab and can be studied
both in vivo and in vitro conditions.

11. CANDIDA TROPICALIS
• Candida tropicalis is a species of yeast in the
genus Candida.
• It is a common pathogen in neutropenic hosts, in whom it
may spread through the bloodstream to peripheral organs.
• For invasive disease, treatments include amphotericin
B, echinocandins, or extended-spectrum triazole
antifungals.
• C. tropicalis is a vegetative cell with the shape from round
to oval ranging from approximately 2 – 10 micrometers.
• A mould exhibits dimorphism forming a single-celled yeast
or so-called blastoconidia which reproduces by simple
budding.

12. • Conidia is the asexual unit that are produced by budding of
the tips or walls of the hyphae.
• Conidia is a type of simple and unicellular body that could
take the form of multicellular cell with different shapes,
sizes, and colors.
• Microconidia is used to refer to small and unicellular
conidia whereas macroconidia refers to large and
multicellular conidia.
• C. tropicalis reproduces asexually by the production of
blastoconidia through budding.
• As blastoconidia increase in number they may elongate in
shape producing structures called the pseudohyphae.

13. OBJECTIVES:
 TO ANALYSE THE MORPHOLOGY OF
CANDIDA ALBICANS AND CANDIDA
TROPICALIS
 TO CONFIRM CANDIDA ISOLATES USING
BIOCHEMICAL TEST (GERM TUBE TEST)
 TO STUDY THE GENETIC DIVERSITY OF
C.TROPICALIS AND C.ALBICANS USING
RAPD MARKERS.

14. MATERIALS REQUIRED
Different isolates of Candida species:
Candida tropicalis Resistant. (CTR)
Candida tropicalis Intermediate. (CTI)
Candida tropicalis Susceptible. (CTS)
Candida albicans Susceptible.(CAS)
Candida albicans Flu Resistant. (CAFR)
COMPONENTS USED
TAE Buffer, Potato Dextrose Agar, Lactophenol Cotton Blue, Human
Serum, Phenol, Chloroform, Isoamylacohol, Agarose Gel, Loading
dye, Milli Q Water, Taq Buffer, Mgcl2, dNTPS, Primers, Template,
Taq Polymerase.

15. MEDIA PREPARATION
PROCEDURE
The potato dextrose dextrose agar was prepared and
autoclaved.
10 plates were prepared and were streaked with different
Candida species isolates.
The plates were kept for 3 days and the growth was observed.

16. RESULT & INTERPRETATION
SL.No. SAMPLE GROWTH
1. Candida tropicalis Resistant Negative
2. Candida tropicalis
Intermediate
Negative
3. Candida tropicalis Susceptible Positive
4. Candida albicans Susceptible Negative
5. Candida albicans Resistant Positive
Candida tropicalis susceptible Candida albicans flu resistant

17. FUNGAL STAINING
The lactophenol cotton blue wet mount preperation is the most widely
used method of staining and observing fungi.
The preparation has three components:
1. Phenol: Kills any living organisms.
2. Lactic acid: It prevents fungal structures.
3. Cotton blue: It stains the chitin in the fungal cells.
REQUIREMENTS
Lactophenol Cotton Blue and Human Serum

18. Candida tropicalis susceptible Candida albicans flu resistant

19. INTERPRETATION
SL.NO. Sample Observation
1. Candida tropicalis
susceptible
The spores are
immature cells.
2. Candida albicans
flu resistant
The hyphae and
septate are
separated.
It is a matute cell.

20. GERM TUBE TEST
PRINCIPLE
Formation of germ tube is associated with increased synthesis
of proteins and ribonucleic acid. Germ tube solutions contains
tryptic soy broth and fetal bovine serum essential nutrients for
protein synthesis. It is lyophilized for stability. Germ tube is one
of the virulence factors of Candida albicans. This is a rapid test
for the presumptive identification of C.albicans.
when Candida is grown in human or sheep serum, at 37 degree
celcius for 3 hours, they forms a germ tube, which can be
detected with KOH films as filamentous outgrowth extending
from yeast cells.
REQUIREMENT
• Tryptic soy broth
• Fetal bovine serum

21. PROCEDURE OF GERM TUBE TEST
0.5ml human serum was taken into a small tube.
Using a Pasteur pipette, touch the colony of the isolated
species and gently emulsify it in the serum.
Incubated the tube at 37°C for 2 to 4 hours.
Transfer a drop of the serum to a slide for examination.
Coverslip and examine microscopically under low and high
power objectives.

22. RESULT & INTERPRETATION
SL.No. SAMPLE GERM TUBE
1. Candida tropicalis
resistant
Negative
2. Candida tropicalis
intermediate
Negative
3. Candida tropicalis
susceptible
Negative
4.
Candida albicans
susceptible
Negative
5.
Candida albicans flu
resistant
Positive

23. MN
CANDIDA
ALBICANS
FLU
RESISTANT

24. SUBCULTURING OF THE MICROORGANISM
• Subculturing describes the transfer of microbes from one
growth medium container, such as broth or agar to another
and allowing the microbes to grow. Subculturing is also
useful in keeping strains alive by transfering them to fresh
growth medium.
• Subculturing allows an analyst to move microbes from one
set of test parameters, such as temperature and media
type to another. This information is useful in microbial
identification, as some species will grow and some will not,
depending on the parameters chosen. He can also keep
cultures alive by subculturing them onto a new growth
medium before the microbes use up all the nutrients in
their growth medium and die.

25. PROCEDURE
100 ml of Potato Dextrose Agar was prepared.
Autoclaved the media and pour into respective labeled
tubes.
Inoculated the different Candida isolates into the broth and
kept it in the incubator of 2 days.

26. DNA ISOLATION
REQUIREMENT
TE Buffer, 10% SDS, Proteinase K, Phenol:
Chloroform: Isoamyl alcohol, isopropanol,

27. DNA ISOLATION
PROCEDURE
Take 1.5ml of the culture and centrifuge it @ 5,000 rpm for
5 minutes.
Discard the supernant and to the pellet add 467 micro litre
of TE Buffer, 30 micro litre of 10% SDS and 3 micro litre of
Proteinase K.
Dissolve the pellet by tapping and incubate the tubes at 37
degree celcius for 1 hour.

28. To the contents add 500 micro litre of Phenol: Chloroform:
Isoamyl alcohol mix well and centrifuge the tubes @ 10,000
rpm for 10 minutes.
Carefully transfer the aqueous layer to the fresh tubes and
add twice the volume of isopropanol.
Incubate the tubes at -20 degree celcius for 1 hour
Centrifuge the tubes @ 10,000 rpm for 10 minutes.
Discard the supernatant and to the pellet add 200
microlitre of 70% chilled Ethenol

29. Centrifuge the tubes @ 10,000 rpm for 10 minutes.
Discard the supernatant, air dry the pellet.
Add 20-50 microlitre of isopropanol to the pellet
and store the samples in -20 degree celcius.

30. QUALITATIVE ANALYSIS OF DNA
AGAROSE GEL ELECTROPHORESIS
Gel Electrophoresis seperates DNA fragments by size in a solid
support medium . DNA samples are pipetted into the wells.
Applications of an electric current at the top (anodal,negative) end
causes the negatively charged DNA to migrate to towards the
bottom (cathodal, negative ) end. The rate of migration is
proportinal to size: smaller fragments move more quickly.
DNA is visualized by including in the gel an interacting dye Ethidium
Bromide. DNA fragments take up the dye as they migrate through
the gel. Illimination with UV light causes the intercalated dye to
fluoresce with a pale pink colour.

31. REQUIREMENT
• Agarose gel, TAE buffer, loading dye

32. PROCEDURE
2% 0f 50ml agarose gel was prepared by adding 1gm of
agarose to 50ml of TAE buffer.
Heated it in the Hot Mantle until it becomes a clear solution.
Pour the gel in the casting tray and keep aside for
solidification.
Remove the comb gently.

33. Mix the isolated DNA with the loading dye and load it
in the wells.
Run the gel for 1 hour under 50 volt.
Observe the bands.

34. OBSERVATION OF GEL
SL.NO. Sample
1 CTI (1)
2 CTI (2)
3 CTR (1)
4 CTR (2)
5 CTR (3)
6 CTS (1)
7 CTS (2)
8 CAS (1)
9 CAS (2)
10 CAS (3)
11 CAFR (1)
12 CAFR (2)
9 10 11 12
1 2 3 4 5 6 7 8

35. QUANTITATIVE ANALYSIS OF DNA
• Quantification of nucleic acids is commonly used in
molecular biology to determine the concentration of DNA
present in the mixture.
• As subsequent reaction or protocols using the nucleic acids
sample of a required particular amount of optical
performance.
• Both DNA and RNA exhibit strong absorbance of UV due to
the presence of conjugated double bonds of the constant
purine and pyramidine bases and these have characteristics
of OD of absorbance maximum at 260 nm which is linearly
related with the concentration of the DNA in the solution
upto the OD value of 2.
• The spectroscopic method is used to check the purity of
DNA.

36. To analyse the quantity of DNA, the absorbance was
take at 260, 280 and 230.
Sl.No Sample OD at 230 OD at 260 OD at 280
1 CTI (1) 0.0428 0.0439 0.0357
2 CTI (2) 0.0447 0.0876 0.0799
3 CTR (1) 0.0138 0.0187 0.0151
4 CTR (2) 0.0070 0.0198 0.0186
5 CTR (3) 0.0037 0.0120 0.0102
6 CTS (1) 0.0105 0.0198 0.0147
7 CTS (2) 0.0019 0.0113 0.0072
8 CAS (1) 0.0123 0.0134 0.0063
9 CAS (2) 0.0071 0.0142 0.0028
10 CAS (3) 0.0124 0.0128 0.0042
11 CAFR(1) 0.0124 0.0131 0.0152
12 CAFR(2) 0.0158 0.0143 0.0058

37. PURITY OF THE DNA
The most common technique to determine DNA yeild and
purity is measurement of absorbance. To evaluate DNA
purity, measure absorbance from 230nm to 320nm to
detect other possible contaminants. The most common
purity calculation is the ratio of the absorbance at 260nm
divided by the reading at 280nm. Good quality will have an
A260/A280 ratio of 1.7-2.0.

38. SL. No. Sample Purity
1 CTI (1) 1.229
2 CTI (2) 1.096
3 CTR (1) 1.238
4 CTR (2) 1.311
5 CTR (3) 1.176
6 CTS (1) 1.346
7 CTS (2) 1.569
8 CAS (1) 0.212
9 CAS (2) 1.692
10 CAS (3) 3.047
11 CAFR(1) 1.861
12 CAFR(2) 2.465
Accordingly, the pure samples that are selected for PCR are:
CTI (1), CTR (2), CTS(2), CAS(2), CAFR(1)

39. DNA CONCENTRATION
DNA concentration is estimated by measuring the
absorbance at 260nm, adjusting the A260 measurement for
turbidity (measurement by absorbance at 320nm),
multiplying the dilution factor, and using the relationship
that an A260 of 1.0 = 50 micro gram per ml.
Concentration =(A260 reading- A320 reading) x dilution
factor x 50

40. SL.NO. Sample DNA conc.
1 CTI (1) 2195
2 CTI (2) 4380
3 CTR (1) 935
4 CTR (2) 990
5 CTR (3) 605
6 CTS (1) 990
7 CTS (2) 565
8 CAS (1) 920
9 CAS (2) 710
10 CAS (3) 640
11 CAFR (1) 655
12 CAFR (2) 715

41. RAPD ASSAY
• RAPD stands for Random Amplification of Polymorphic
DNA.
• It’s a type of PCR, but the segments of DNA that are
amplified are random.
• RAPD markers are decamer ( 10 necleotides long) DNA
fragments from PCR amplification of random segments of
genomic DNA with single primer of arbitraary nucleotide
sequence and which are able to differentiate between
genetically distinct individuals although not necessarily in
areproducible way.
• It is used to analyse the genetic diversity of an individual
by using random primers.
• RAPD requires only one primer for amplification.

42. HOW IT WORKS ?
• Unlike traditional PCR analysis, RAPD does not require any
specific knowledge of the DNA sequence of the target
organism: the identical 10-mer primers will or will not
amplify a segment of DNA, depending on positions that are
complementary to the primers' sequence.
• For example, no fragment is produced if primers annealed
too far apart or 3' ends of the primers are not facing each
other.
• Therefore, if a mutation has occurred in the template DNA
at the site that was previously complementary to the
primer, a PCR product will not be produced, resulting in a
different pattern of amplified DNA segments on the gel.

43.  RAPD-PCR reaction was carried out using 8 primers namely
. The reaction mixture is listed out in the table 1. The
amplification reaction was carried out as mentioned in
table 2 on Lark mastercycler. The amplified products were
resolved by electrophoresis on 2% agarose gel containing
ethidium bromide along with ladder DNA as a standard
molecular weight marker. The gel was visualized under UV
transilluminator and photographed in a gel documentation
system.

44. LIST OF PRIMERS TO BE USED:
SL.No. Primer Name Sequence Name Sequence (5’-3’)
1. Primer 23 OPO 7 GACCACTGAC
2. Primer 29 OPN 11 TCGCCGCAAA
3. Primer 30 OPN 12 CACAGACACC
4. Primer 31 OPN 13 AGCGTCACTC
5. Primer 32 OPN 14 TCGTGCGGGT
6. Primer 11 OPB 19 ACCCCCGAAG
7. Primer 9 OPB 12 GTGATCGCAG
8. Primer 19 OPO 5 CCCAGTCACT
NOTE:
The information is given by INDIGENOUS DNA PVT. LTD
Name of client: SYNERGY SCIENTIFIC SERVICES.
No. 11/61-A, Ayisha towers,III floor, Vandikaran street, Maduvinkarai, Gunidy,
Chennai

45. Table 1: REACTION SET UP FOR RAPD PCR
SL.No. Components Volume for 1X
reaction(in µl )
Volume for 6X
reaction (in µl)
1 Milli Q Water 11.63 69.78
2 Taq Buffer 2.0 12.00
3 Mgcl2 0.2 1.2
4 dNTPs 2.0 12.00
5 Primer 2.67 16.02
6 Template 1.0 -
7 Taq Polymerase 0.5 3.0

46. • TABLE 2: TEMPERATURE PROFILE FOR RAPD
REACTION
Step Temp Time No. of cycles
Initial
Denaturation
95°C 3mins 1
Denaturation 95°C 45secs 40
Primer Annealing 37°C 1min
Extension 72°C 1min 20secs
Final Extension 72°C 8mins 1

47. DATA ANALYSIS
• Amplification profiles of five samples of Candida
species were compared with each other and bands
of DNA fragments scored manually depending on
the presence (1) or absence (0) of band.

48. RESULT OF PCR
DNA of Candida species on 2% agarose gel
The genetic relationship between Candida species samples
collected were analysed using RAPD markers. Two primers
generated informative and easily scorable band profiles
with DNA fragments . The populations were highly
differentiated by their own genetic distance in the UPGMA
dendrogram based on Nei’s genetic distance.

49. M- 1kb Ladder
A- Primer 23
1A- CTS
2A- CTI
3A- CAS
4A- CTR
5A- CAFR
M 1A 2A 3A 4A 5A
PCR AMPLIFICATION RESULT OF OPO7 IN 2%
AGAROSE GEL

50. M- 1kb Ladder
B-Primer 29
1B- CTS
2B- CTI
3B- CAS
4B- CTR
5B- CAFR
PCR amplipication result of OPN 11 in 2%
agrosegel

51. Dendrogram representing the genetic diversity of Candida
isolates using primer 23 and 29

52. CONCLUSION
The Isolates Candida tropicalis susceptible and Candida
albicans Flu Resistant are similar, but it is not true. Where
as Candida tropicalis intermediate and Candida tropicalis
resistant are closely related with distance of 0.577, it is
true because they are only of the same species. But isolate
Candida albicans Susceptible is related with isolates
Candida tropicalis susceptible, Candida tropicalis
intermediate, Candida tropicalis Resistant and Candida
albicans Flu Resistant at a distance of 0.707, but is unable
to differentiate between Candida albicans and Candida
tropicalis. For further diversity, large number of primers
should be used.

53. DISCUSSION
 Molecular analysis was undertaken using RAPD generated by eight
primers. This technique enables us to examine the particular
sequences or type of sequences and can thus be taken as
representative of the genome.
 RAPD was used to analyze the genomic variability of the five isolate
obtained from patients. We were particularly interested in
differentiating the isolates for making a marker against the
particular sequence.
 This technique produces a profile of bands that allowed the
identification of intra and inter specific polymorphisms among
isolates. Additionally it differentiated Candida strains isolated from
different patients.

54. In the present study, we observe that RAPD technique
was able to point out clearly the genomic viability within
the Candida isolates. The identification of specific markers
was done.
According to the result obtained, RAPD assay can be
considered important tool to identify as well as study
inter and intra specific genetic variability among Candida
isolates. Moreover this technique allowed the
identification of distinct strain of Candida.

55. THANK
YOU