Microbiology Semester 2 Week 2

        Assalamualaikum and hi everyone... how are you all? I hope everyone will be fine as well... On Tuesday, in microbiology class as usual Dr Wan was taught us more about the fungi. Before that, Dr Wan was checked our flowchart about the isolation of the fungi. If any mistakes, she told us to change and correct it. For an hour, we did again the flowchart that Dr Wan asked us to correct it include the references. Next, Dr Wan taught us about the fungi in more details. Basically, Fungi is a eukaryotic spore-bearing organisms. Fungi does not have chlorophyll and it can reproduce by both sexually and asexually. It also is the absorptive nutrition. Fungi are divided into three which are true fungi, fungi imperfecti and fungus-like. Example of the Fungus-like are slime molds and water molds.

     On friday, Dr Wan was gave a group task to find information about how to identify eukaryotes? For my group, How to identify fungi because my group are fungi so we have find about fungi. Some groups are find about protozoa, archaea, virus, algae and bacteria. My group was found about Molecular identification and control of some pathogenic Fusarium species isolated from maize in Egypt by Izzati, Detection, Identification, and Distribution of Fungi in Bronchoalveolar Lavage Specimens by Use of Multilocus PCR Coupled with Electrospray Ionization/Mass Spectrometry by Adriana, Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia
species by Adriana, Molecular identification of pathogenic fungi by Nadhirah, Molecular identification of fungi isolated from coastal regions of Red Sea, Jeddah, Saudi Arabia by Amira, Identification of fungi from dairy products by means of 18S rRNA analysis by me, Identification of fungi in the Gaeumannomyces-Phialophora complex by RFLPs of PCR-amplified ribosomal DNAs by me and Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis systems by Luqman. If you want to study more about the how to identify fungi you cal also visit the page and the links are at the below.

Molecular identification and control of some pathogenic Fusarium species isolated from maize in Egypt by Izzati
https://www.researchgate.net/publication/272167634_Molecular_identification_and_control_of_some_pathogenic_Fusarium_species_isolated_from_maize_in_Egypt

Detection, Identification, and Distribution of Fungi in Bronchoalveolar Lavage Specimens by Use of Multilocus PCR Coupled with Electrospray Ionization/Mass Spectrometry by Adriana
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536260/

Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia
species by Adriana
https://www.researchgate.net/publication/257748591_Molecular_phylogenetic_identification_of_endophytic_fungi_isolated_from_three_Artemisia_species

Molecular identification of pathogenic fungi by Nadhirah
https://www.ncbi.nlm.nih.gov/pubmed/18063605

Molecular identification of fungi isolated from coastal regions of Red Sea, Jeddah, Saudi Arabia by Amira

https://www.sciencedirect.com/science/article/pii/S1815385216300384

Identification of fungi from dairy products by means of 18S rRNA analysis by me
https://www.sciencedirect.com/science/article/pii/S0168160501005876

 Identification of fungi in the Gaeumannomyces-Phialophora complex by RFLPs of PCR-amplified ribosomal DNAs by me
https://www.sciencedirect.com/science/article/pii/S0953756209801895

Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis systems by Luqman
https://www.ncbi.nlm.nih.gov/pubmed/14653513


   

Microbiology Semester 2 Week 1

        Assalamualaikum and hi everyone... how are you all? I hope everyone will be fine as well... long time no post anything in this blogspot.... This is the first week of microbiology class in semester 2.... semester 2 is more detail than the first semester because first semester is just a introduction about the microorganisms. Semester 1 is over ready still 7 semester more to complete my bacelor in science microbiology in honors. In semester 2, for microbiology I have two classes per week on tuesday and friday. This semester I have three lecturer's that going to teach me which are Dr Wan, Dr Asilah and Dr Suriana. First day, in microbiology class as usual Dr Wan was gave some briefing about the topics that we cover on this semester and also about the assignment "Pecha Kucha". She also was briefing about the percentage of the test, assignment and quizzes.

        On Friday, Dr Wan asked us to do flowchart about how to isolate fungi in groups. My group did on isolation of fungi from tropical soil. The purpose of isolate of fungi from tropical soil is to isolate secondary metabolites such as antibiotic, enzyme, protein and so on.

Fungi produced two types of metabolites

Primary metabolites
Fungi produced primary metabolites for their proper growth, reproduction and their proper development.

Secondary metabolites
While secondary metabolites fungi produce for their own defense, to grow in the same medium and to killed other microbes in the same medium.

Name of the culture medium used for fungi growth 

• Brain heart infusion agar
• Czapek yeast agar
• Czapek yeast broth
• Inhibitory mold agar
• Mycosel/Mycobiotic agar
•  Potato Dextrose agar
• Sabouraud's heart infusion agar
• Bannerot synthetic media agar
• Potato flake agar
• Water agar  

ISOLATION OF FUNGI FROM TROPICAL SOIL

• Prepare Potato Dextrose Agar (PDA)
• Dig earth surface at 10-12 cm depth and collect soil.
• Perform serial dilutions.
• Inoculate sample using spread plate method on the PDA.
• Incubate the agar plate in inverted positions at 28⁰C for 48 hours.
• Subculture the single colony into new PDA to isolate pure culture.

Isolation of fungi in bread mold

https://www.ijcmas.com/vol-4-1/Unachukwu%20M.N%20and%20Nwakanma%20C..pdf

Isolation of fungi from seawater

www.mdpi.com/2309-608X/3/3/36/pdf

FLOW CHART OF ISOLATION GUAVA’S TREE DISEASE

1.       Samples were collected from infected tissues to ascertain the association of different pathogens. The samples consisting of bark at collar region, twigs and soil were collected. Trees showing declining symptoms were selected and samples of roots, soil, bark at collar region, and leaves of healthy as well as diseased plants were collected.

2.       After that, blench all the disease’ parts by using mortar into a powder. The infected samples (in form of powder) are mix with distilled water.

3.       Infected samples are plated on PDA by using the serial dilution technique. The plate that need to be inoculated are in a several dilution factor by using spread plate (NOTE: try to inoculate the sample in higher diluting factor).

4.       Incubated all the plates in incubator at 27 °C for 3‒7 days and data was recorded. The fungi which colonized on these pieces was purified and identified on the basis of their morphological characters.

5.       Cut the small pieces of fungus by using scalpel, that differ by the morphology into a new plate. Place it on the middle plate and incubated the plate again in suitable temperature again (27 °C) for another week. For the type of fungus that bring the disease, Botryodiplodia theobromae, the mycelial growth of isolates was classified as fluffy or depressed, uniform to irregular and cottony white turning to black.

6.       After a week, observe the morphology of fungus and record in your Results                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   

Microbiology Week 14

Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is fourteenth week of my microbiology class. In microbiology, we study about the nutritional types of microorganisms.

2 major nutritional concern:
a.Source of energy
b.Source of carbon

Energy sources
• phototrophs: light as primary energy source
• chemotrophs: inorganic and organic compunds as primary energy source (from redox reactions)

Carbon sources
• Autotrophs (self feeders): CO2 – lithotrophs
• heterotrophs (feeders on others): organic carbon – organotrophs

Combine the energy and carbon sources:
• Photoautotrophs
• Photoheterotrophs
• Chemoautotrophs
• Chemoheterotrophs

Photolithoautotrophy
- Photolithotrophic autotrophs or photoautotrophs
- Source of energy: Light Source of C: CO2 Source of e-: reduced inorganic molecules e.g. Algae and cyanobacteria: H2O Purple and green sulfur bacteria: H2, H2S and elemental S

Photoorganoheterotrophy
- Photoorganotrophic heterotrophs
- Source of energy: Light Source of C: Organic C molecules Source of e-: Organic molecules
- Common inhabitants of polluted lakes/streams

Chemolithoautotrophy
- Chemolithotrophic autotrophs
- Source of energy: Oxidation of organic/inorganic compounds Source of C: CO2 Source of e-: Reduced inorganic molecules (iron, N, S)
- Contribute to chemical transformations of elements in the ecosystem

Chemoorganoheterotrophy
- Chemoorganotrophic heterotrophs or chemoheterotrophs
- Source of energy: Oxidation of organic/inorganic compounds Source of C: Organic C molecules Source of e-: Organic molecules
- Same organic nutrient à satisfy all requirements
- Most pathogenic m/os

Mixotrophic
- Alter metabolic patterns in response to environmental changes
- E.g. Purple nonsulfur bacteria Photoorganoheterotroph
    – absence of O2 Chemoorganoheterotrophs
    – presence of O2 The term mixotroph can describe organisms (usually algae or bacteria) capable           of  deriving metabolic energy both from photosynthesis and from external energy sources. These         organisms may use light as an energy source, or may take up organic or inorganic compounds.

Photoautotroph
• Photosynthetic bacteria, algae, cyanobacteria and green plants.
• obtain energy from photophosphorylation and fix carbon from CO2 via the Calvin-Benson cycle to synthesize organic compounds.
• photosynthetic bacteria:
– oxygenic phototrophs, produces O2 : cyanobacteria.
– anoxygenic phototrophs, do not produce O2, anaerobic condition: green sulfur bacteria (eg. Chlorobium) and purple sulfur bacteria (Chromatium)
• Green sulfur bacteria
– Chlorobium
– Use sulfur (S), sulfur compounds (H2S) or hydrogen gas (H2) to reduce CO2 and form organic compounds.
• Purple sulfur bacteria
– Chromatium
– Also use sulfur, sulfur compounds or H2 to reduce CO2.
– They are distinguished from the green sulfur bacteria by the location of their bacteriochlorophyls, location of stored sulfur and ribosomal RNA

Photoheterotrophs
• use light as an energy source and an organic compound for their carbon source or electron donor
• They are anoxygenic
• purple nonsulfur bacteria (Chloroflexus)
• green non sulfur bacteria (Rhodopseudomonas)

Chemoautotrophs
• use inorganic compounds as their energy source and CO2 as their carbon source
• Inorganic sources of energy:
– H2S : Beggiatoa
– S : Thiobacillus thioxidans
– NH3 : Nitrosomonas
– NO2 - : Nitrobacter
– H2 : Hydrogenomonas
– Fe2+ : Thiobacillus ferrooxidans
• energy stored in ATP - produced by oxidative phosphorylation

Chemoheterotrophs
• use complex organic molecules as their carbon and energy sources
• The energy source and carbon source are usually the same organic compound (glucose)
• most bacteria and all fungi, protozoa and animals
– saprophytes: live on dead organic matter
– parasites: derive nutrients from living host

          

Microbiology Week 13

Assalamualaikum and Good morning.... How r you everyone? I hope everyone will be fine as well. 😊😊😊 This is thirteenth week of my microbiology class. In microbiology, we study about the microbial growth.

Growth vs. Tolerance
 – Growth - referring to the number of cells, not the size.
 – Tolerance - survive under conditions in which they cannot grow.
 – The suffix “-phile” describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow.
 – E.g. a “thermophilic bacterium”, “thermotolerant bacterium”

The requirements for growth
 • Physical requirements
 - light, temperature, pH, water activity and osmotic pressure.
 • Chemical requirements
 - electron donor (C, N, S, P, K)
 - electron acceptor (O2 - , NO3- , SO4 2-, CO3 - , Fe3+) - micronutrients (vitamins, amino acids, trace minerals)

Trace elements - e.g. Fe, Mg, Mn, Cu, Zn …
- Microorganism dependant
- Important for enzyme function
- part of enzyme and cofactors
- catalysis of reactions
- maintenance of protein structure
- Organic growth factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines

Special growth factors
- Specifically needed for growth of certain m/os
- e.g. Legionella pneumophila (Legionaires’ disease)
 ~ absolute requirement for a.a. L-cystein and iron
 ~ in nature
 ~ provided by algae and amoebas

Aseptic technique
 Definition Aseptic technique refers to carrying out a procedure under controlled conditions in a manner that will minimize the chance of contamination. Contaminants may be introduced from the environment, equipment and supplies, or personnel.

 Aseptic Technique
 • Sterile Hood - All manipulations must be carried out in a sterile cabinet – Turn the UV light off (Should ordinarily be on) – Open the cabinet – Wipe down with disinfectant (70% ethanol or 40% isopropyl alcohol)
 • Bring materials into the hood
 • Light up the flame or gas
 • Begin your work
 • Flame all caps and lids
 • Tightly close all boples and caps
 • Remove materials from the hood
 • Turn off gas
 • Wash the hood surface
 • Turn the UV light on to disinfect

Culturing Microorganisms
 • There are two basic culture techniques used in microbiology:
 1. Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.
~ Liquid medium is best when you want to rapidly increase the concentration of the organism or when you want to grow motile cells.
 2. Culture Plates: Liquid medium is solidified using agar (agarose) and poured as a thin layer in the bopom of a culture dish (also some=mes called petri plate)
 ~ Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate individual colonies from environmental samples

Culturing bacteria
 • Culturing bacteria in the laboratory present two problems:
 –To obtain pure culture
 –To use suitable medium

Pure culture
 • Is a population of identical cells originating from a single cell.
 • Pure cultures are obtained by working in aseptic environments.

Anaerobic Environments
 • Reducing Compounds
 – Thioglycholate
 – Cystein
 – Anything with – SH
 – Must Use Indicator
 • Gas Pack (anaerobic generating kit)

Anaerobic Incubation
 • Anaerobic Jar
 – Impermeable to Oxygen
 • Catalyst
 – Platinum or Palladium
 – In Lid or on Gas Pack
 • Gas Pack
 – Uses Oxygen and Replaces with Carbon Dioxide

New technique to provide anaerobic environment
 • Oxyrase
 – Reduces oxygen to water
 – Respiratory enzyme derived from the plasma membranes of certain bacteria
 – Added into growth medium
 • Avoid the need for more cumbersome apparatus

Culture media
 CULTURE MEDIUM
- A nutrient material prepared for the growth of microorganisms in a laboratory.
 INOCULUM
 - Microbes that are introduced into a culture medium to initiate growth.
 CULTURE
- The microbes that grow and multiply in or on a culture medium.
 STERILE
- No living microbes

Types of Culture Media
 • Chemically defined vs. complex media
 – Chemically defined media
 • The exact chemical composition is known • e.g. minimal media used in bacterial genetics experiments
 – Complex media
 • Exact chemical composition is not known
 • Oten consist of plant or animal extracts, such as soybean meal, milk protein, etc.
 • Include most routine laboratory media, e.g., tryptic soy broth.
 • Liquid (broth) vs. semisolid media
 – Liquid medium
 • Components are dissolved in water and sterilized
 – Semisolid medium
 • A medium to which has been added a gelling agent
 • Agar (most commonly used)
 • Gelatin
 • Silica gel (used when a non-organic gelling agent is required)
 • Selective media
 – Contain agents that inhibit the growth of certain bacteria while permiting the growth of others
 – used to isolate specific organisms
 • Differential media
 – Contain indicators that react differently with different organisms (for example, producing colonies with different colors)
 – Used in identifying specific organisms
 • Enrichment media
– The enrichment media will increase the small numbers of desired bacteria to detectable level.
– One type of bacteria present in small numbers while the other type present in much larger numbers. – The enrichment medium is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe but not others.