How to identify a mold? What to do and not to do? We delve into the fascinating world of fungal identification, exploring traditional and molecular methods to isolate, characterize, and document fungi. Let's begin. Isolation is key to understanding fungi. It involves obtaining a pure culture. Here's what you should do: Sample Collection: Collect specimens aseptically, preserving the integrity of the sample. Selective Media: Utilize appropriate media, adjusting pH and nutrients for fungal growth. Incubation: Maintain optimal conditions—temperature, humidity—for fungal growth. Subculture: Transfer colonies to obtain pure cultures. What not to do: Avoid contamination—sterile technique is crucial throughout. Lactophenol cotton blue staining helps observe fungal structures. How to perform it: Preparation: Make a smear from the culture on a slide. Application: Add a drop of lactophenol cotton blue, allowing it to permeate. Examination: Observe under a microscope for morphology and staining patterns. What not to do: Overstaining can distort structures—use appropriate quantities. Traditional methods involve observing morphology, growth patterns, and biochemical tests. Molecular techniques use DNA analysis. Traditional: Observe colony morphology, growth rate, and perform biochemical tests. Molecular: PCR amplification and DNA sequencing for species-specific identification. What not to do: Rely solely on one method—combine both for accurate identification. Proper documentation ensures accurate records and reproducibility. Note-Taking: Detailed records of isolation, staining, and observations. Photography: Capture microscopic and macroscopic features for reference. Data Compilation: Store all data systematically for future analysis. What not to do: Incomplete documentation—record every step meticulously.

1. Eukaryotic Microbiology
Lab – isolation of fungi from
1.Soil
2.Surface of leaf
3.Within the leaf
3 basic qualities of mycologist
1. Producing clean control
2. Producing and maintaining pure cultures
3. Producing countable fungal colonies

2. Step 1 Step 2 Step 3
Visualization of the
experiment before
entering the lab
Execution of the
experiment
Cleaning before
leaving the lab

3. Documenting
fungal
morphologies

4. Record the source of isolation

5. Fungal characterization
Macro-morphological characterization
1. Fungal colony
characteristics
2. Reverse plate venations
3. Spore color
4. Pigment production
Micro-morphological characterization
1. Mycelial structure
2. Hyphal nature
3. Spore bearing structure
4. Spore structure
Molecular characterization
1. ITS gene
sequencing
2. Protein profiling

6. Mother plates

7. Front view Reverse view Front view Reverse view
Day 1
Day 2
Day 3
Day 4

8. Front & reverse plate

13. Penicillium expansum
Czapek yeast extract agar
Malt extract agar
Potato dextrose agar
Yeast extract sucrose agar

18. Spore structure

19. Broth morphology

20. Soil fungi

21. Isolation of endophytes

22. The isolation of specific endophytic fungal
species is affected by the concentration of
surface sterilizing agents and the duration
of treatment

23. Reverse
Front

24. 4X objective
Bacterial biomass
Fungal biomass
hyphae
40X
Is this an endophyte? NO
Growth by tip extension
branching
Endo-bacteria
emerging out
from the leaf
Endobacteria
growing within
the leaf
Growth of
endophytic
bacteria
under the leaf
surface
Inoculated leaf
tissue under 40X
Need for clean control

25. Endophyte
Not an endophyte

26. Maximum of 4
implants could be
inoculated in a plate

29. Endophytic bacteria

30. Endo-phytic basidiomycota

31. Slide culture technique

33. Preserving molds in distilled water – as agar buttons

34. Reviving from agar buttons

35. Sample collection
and documentation
Plating onto PDA –
mother plate
Pure culture in PDA
1. Every day documentation of
front and reverse plate
2. Measure the colony size
twice a day (12 h interval)
Lacto-phenol cotton blue staining,
document
1. Mycelial structure
2. Hyphae
3. Spore bearing structure
4. Spore structure
Eukaryotic Microbiology work plan – template
Observing results and
documentation
Slide culture technique
Observe the results every
day
Preservation
1. Glycerol stock
2. Agar buttons in
dis. H2O
Broth culture
characterization &
documentation
Anti-fungal assay
Mold DNA isolation &
gel electrophoresis
PCR: ITS gene
amplification
Stock re-viability test
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36. Isolation of entomo-pathogenic fungus