Oxidase Test

Oxidase enzymes play a vital role in the operation of the electron transport system during aerobic respiration.  Cytochrome oxidase catalyses the oxidation of a reduced cytochrome by molecular oxygen (O₂), resulting in the formation of H₂O or H₂O_2.  Aerobic bacteria as well as some facultative anaerobes and microaerophiles, exhibit oxidase activity.  The oxidase test aids in differentiation among members of the genera Neisseria and Pseudomonas, which are oxidase positive, and Enterobacteriaceae, which are oxidase negative.

Aim

To distinguish among groups of bacteria on the basis of cytochrome oxidase activity.

Principle

The ability of bacteria to produce cytochrome oxidase can be determined by the addition of the test reagent, p-aminodimethylaniline oxalate, a reagent which serves as an artificial substrate, donating electrons and thereby becoming oxidized to a blackish compound in the presence of the oxidase and free oxygen.  The dark coloration is indicative of cytochrome oxidase production and represents a positive test.  No color change is indicative of the absence of oxidase activity and is a negative test.

Requirements

24 hr nutrient broth cultures of species Bacillus, Staphylococcus, Streptococcus, Pseudomonas and E.coli.

Oxidase strips containing p-aminodimethylaniline oxalate, Bunsen burner, glass rod, glass marking pencil etc.

 Procedure

1. Remove oxidase strips from the container and place in a petridish.
2. With the help of a clean glass rod / plastic loop or platinum wire pick a colony from 24 hrs growth of      the test organism and rub over the filter paper.  Do not use nichrome wire.
3. Observe the color change within 5-10 sec. 

 Observations

Pseudomonas sp. produced an intense deep blue color within 5-10 sec indicating a positive result.   Bacillus, Staphylococcus, Streptococcus and E.coli produced no change in color.

 Result

Pseudomonas sp. is oxidase positive whereas Bacillus, Staphylococcus, Streptococcus and E.coli are oxidase negative.

(Image courtesy: orbitbiotech.com)

Animal Cell Culture as a Substitute for Animal Experiments- Dr C R Meera

 

Animals are extensively used for research purposes. They are mainly used for efficacy testing and toxicological screening of drugs for different infectious and non-infectious diseases and for screening of carcinogens. Animals are also sacrificed for medical and surgical experiments. They are also widely used for production of vaccines, antibiotics etc. The number of animals used for experiments have increased considerably with advancement in medical technology & research. Millions of experimental animals are exploited or sacrificed all over the world every year.  Various animals are used for this purpose which include rodents (mice, rats, hamsters, rabbits, guinea pigs), fishes (zebra fish, trout), birds (mainly chicken), amphibians (xenopus frogs), primates, dogs, cats etc. Many animals die during experiment and others are sacrificed at the end of experiments. The pain, distress and death experienced by the animals during scientific experiments have been a debating issue for a long time. Various acts and laws have been passed to bring the control over unethical use of animals and minimize the pain to animals during experimentation. Regulations put forward by the Animal Ethics Committee have to be strictly followed for conducting animal experiments.

 

Animal cell culture, growing cells in dishes is an excellent alternate for animal experiments. It has many advantages over animal experiments.

1. Cost and space required are more in animal experiments. Cost of animal experiments are high as breeding, maintenance and care of animals are to be done according to Government rules. Animals have to be maintained in specific breeding centers occupying large areas associated with the research centers. Cell culture is less expensive compared to animal experiments and cells can be grown in small containers. Space consumed for cell culture lab is also very less or even we can work with cell lines on a bench top.
2. Animal experiments are difficult to perform and require skilled/trained manpower is required to handle the experiments. Cell culture techniques are easy to perform and less manpower required.

3.       In animal experiments, the number of animals required for a single experiment is many. But in cell culture, many experiments can be done using one animal by dividing the tissue into different experimental groups. Thus multiples of the test can be easily performed.  

   E.g.: Almost all cosmetics, drugs and chemicals are tested for their toxicity and efficacy before commercialization. For example, irritancy test. To check the irritancy of cosmetics/chemicals previously Draize test was used, which requires animals (mainly rabbit). It is very painful and every time a new animal is used. The procedure involves applying 0.5 mL or 0.5 g of a test substance to the eye or skin of a restrained, conscious animal, and then leaving it for set amount of time before rinsing it out and recording its effects. The animals are observed for up to 14 days for signs of erythema and edema in the skin test, and redness, swelling, discharge, ulceration, hemorrhaging, cloudiness, or blindness in the tested eye.  Cell culture replaced it with Corneal organ culture. The bovine cornea is cultured up to three weeks in laboratory and various analytical methods are used to evaluate the toxicological effect of test chemical irritancy in vitro.              Live animals and embryos are used to study effects of some compounds on embryo development. In vitro embryonic stem cell culture test helps to reduce the number of live embryo used and the compounds which are toxic toward developing. 

4. Another advantage of cell culture is time. Animals experiments have time consuming lengthy protocols and takes considerable time. It usually takes a whole day to do one experiment. Organ or tissue culture experiments are of shorter duration compared animal experiments and done with multiple samples at one time.

5. .   Tissue culture is having controlled physiochemical environment (pH, temperature, osmotic pressure, O₂, CO₂ tension etc.) as well as physiological conditions within the system which cannot be controlled in animal experiments. Hence results of tissue culture are more reliable than animal experiments.

6.     Cell cultures are more suitable for screening the efficacy or toxicity of drugs, carcinogens and chemicals.  In cell culture, the compound to be screened is cultured with isolated organ, tissue or cells. In such systems, it is easy to understand the response of cell line towards the drug. In the whole animal, it is not easy to determine the specific effect of a particular drug. Because, it is possible that known or unknown endogenous substances in the animal body may be interacting with the exogenous material being tested.

7.         Also less reagents are required in cell culture which are readily available to target cells. In animal experiments, 90% injected chemical is lost by excretion and distribution to tissues other than target cells. In cell culture, cells are exposed directly to the reagent at a lower and defined concentration and with direct access to the target cells.

8.     In vitro cell culture is a good way to screen the compounds at preliminary stages. For example, use of the human hepatocyte culture gives the information about how a drug would be metabolized and eliminated from the body. Such experiments help to eliminate unsuitable compounds from further studies and thus minimizes the use of animals in further experiments.

9.       Tissue samples collected from animals for experiments are invariably heterogeneous. But cultured cell lines, after one or two serial passages assume a homogeneous or uniform constitution which reduces the need for statistical analysis of variance. 

Asexual Reproduction in Fungi- Dr C R Meera

 Asexual Reproduction in fungi

Fungi reproduce by both sexual and asexual means. 

• Yeasts which are unicellular fungi reproduce by budding which is an asexual process. Eg: Cryptococcus neoformans. Some yeasts like Schizosaccharomyces pombe reproduce by fission instead of budding and thereby two identically sized daughter cells are produced. Schizosaccharomyces pombe is a facultative sexual organism and undergoes sexual reproduction under high-stress conditions such as nutrient starvation. In this condition yeast haploid cells will die. However, diploid cells which are formed by conjugation undergo sporulation, entering sexual reproduction (meiosis) and producing a variety of haploid spores, which can go on to mate (conjugate), reforming the diploid.

• Yeast like fungi reproduce asexually by  budding and fission.

• Molds, the filamentous fungi reproduce by both sexual and asexual methods.

• Fungi imperfecti, also called deuteromycetes or hyphomycetes, is a provisional group of fungi whose sexual phases have not been identified.  

1. Asexual Reproduction

Asexual reproduction is also known as somatic or vegetative reproduction. In asexual reproduction there is no union of  nuclei, sex cells or sex organs of two different cells. Daughter cells arise from the single parental cells.  Asexual methods of reproduction include

1. Fission

2. Budding

3. Fragmentation

4. Spore formation (Asexual spores)

   

1. Fission

In fission, the parent cell elongates and divides transversely into two daughter cells of identical size. First, the nucleus divides which is known as Karyokinesis, followed by the division of the cytoplasm and wall formation, known as cytokinesis. In this process, initially the replicated DNA molecule attaches each copy to a different part of the cell membrane. The original and replicated genomes are pulled apart and separated as the cell elongation takes place prior to cell division. All daughter cells produced by fission are genetically identical, meaning that they have the same genetic material. Unlike the processes of mitosis and meiosis used by eukaryotic cells, binary fission takes place without the formation of a spindle apparatus on the cell. Like in mitosis (and unlike in meiosis), the parental identity is preserved.

2. Budding

Budding is the process in which somatic cells produce a small bud like out growth which develops into a new individual. In this method daughter cells pinch or bud from the parental cell and are smaller than the mother cell. Under optimal conditions, yeast cells divide as rapidly as once every 90 min through a process of budding.

3. Fragmentation

In fragmentation, disjoining of hyphal cells take place and each cell develops into a new organism. Disjoined single cells act as the spores and germinate to produce hyphae and mycelium. Thus from each fragmented cell, a new organism arises. Spores produced by fragmentation are called Oidia or Arthrospores or Thallospores (Eg: Erysiphe).

4. Spore formation (Asexual spores)

Asexual spores are produced from single parental cell and their function is to disseminate the species. Fungi that produce more than one type of spores are called pleomorphic or polymorphic fungi as they can exist in different morphological forms. 

Spores are produced at the tip of special hyphae called Sporophores. Name of this special hyphae changes according to the type of spore produced on it.

For Eg: Sporophores that produce Sporangiospores are called Sporangiophores.

    Sporophores that produce Conidiospores are called Conidiophores.

Sporophores bear spores either packed inside special sac-like structures called Sporangium (Pl. Sporangia) or at the terminal ends.  If spores are produced inside the sporangia, they are called endogenous spores and if the spores develop exogenously on the terminal ends of sporophores, they are called the exogenous spores. The sporangia may be terminal or intercalary in their position.

Different types of asexual spores are produced by fungi. They include:

a)Sporangiospores

b)Conidiospores or conidia

c)Oidia (Arthrospores/Thallospores)

d)Chlamydospores

e)Blastospores

a)Sporangiospores   

Sporangiospores are single celled spores produced inside the special sac-like structures called Sporangium (Sporangia Pl.). Sporangia are produced at the tip of  special hyphae called Sporangiophores. A sterile dome-like structure at the tip of a sporangiophore or within a sporangium is called Columella. A funnel-shaped swelling of a sporangiophore, immediately below the columella, can be seen in some fungi and are called Apophysis.

Sporangiospores can be motile or nonmotile. Non-motile sporangiospores are called Aplanospores. Motile ones are called Zoospores and their motility is due to the presence of flagella.

b)Conidiospores or conidia (Conidium)

Conidia are exogenous spores formed at the tip or side of hyphae. They can exist in two forms such as Microconidia and Macroconodia. Microconidia are small single celled conidia whereas Macroconidia are large multi celled spores.

C & d)Oidia/ Oidium (Arthrospores/Thallospores) and Chlamydospores

Both Oidia and Chlamydospores are produced by the disjoining or fragmentation of vegetative hyphae. In oidia formation, hyphal cells simply disjoin from the apical regions and each fragmented cell acts as spore, giving rise to new fungi. In the case of Chlamydospores, cells become enveloped by a thick wall before hyphal fragmentation. These thick walled, single celled Chlamydospores are highly resistant to the adverse conditions and can be terminal or intercalary.

e) Blastospores

Blastospores are spores formed by budding. It is also known as a blastoconidium (pl. blastoconidia). An example of a fungus that forms blastospores is Candida albicans.

Other means of asexual reproduction in fungi include formation of Sclerotia and Rhizomorphs. These asexual methods are usually used to overcome unfavourable environmental conditions. 

The sclerotia are resistant and perennating bodies. Each sclerotium is a cushion-like structure of compact mycelium. They survive for many years.  They give rise to new mycelia on the approach of favourable conditions.

Rhizomorphs are rope-like modified mycelium, also resistant to unfavourable conditions and give rise to new mycelia even after several years on the approach of favourable conditions.

Youtube Link: https://www.youtube.com/watch?v=yX7Ba5VCI9w

Triple Sugar-Iron Agar Test-Dr C R Meera

 

The Triple Sugar-Iron (TSI) agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish the Enterobacteriaceae from other gram negative intestinal bacilli.  This differentiation is made on the basis of differences in carbohydrate fermentation patterns and hydrogen sulphide production by the various groups of intestinal organisms. Some bacteria liberate sulfur from sulfur containing aminoacids or other sulfur containing compounds.  The sulfur is used as final hydrogen acceptor leading to the formation of H₂S. 

Aim

To differentiate among members of the Enterobacteriaceae; and also between the Enterobacteriaceae and other groups of intestinal bacilli.

Principle

The TSI agar slants contain lactose (1%), sucrose (1%) and glucose (0.1%) to facilitate observation of carbohydrate utilization patterns. The acid-base indicator phenol red is also incorporated to detect carbohydrate fermentation that is indicated by a change in color of the medium from orange-red to yellow in the presence of acids.  The slant is inoculated by means of a stab and streak procedure.  Following incubation, the fermentative activities of the organisms are determined as follows:

1.      Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the agar butt): Only glucose fermentation has occurred.  The organisms preferentially degrade glucose first.  Since this substrate is present in minimal concentration, the small amount of acid produced on the slant surface is oxidized rapidly.  The peptones in the medium are also used in the production of alkali.  In the butt the acid reaction is maintained because of reduced oxygen tension and slower growth of the organisms.

2.      Acid slant (yellow) and acid butt (yellow) with or without gas production: Lactose or sucrose fermentation has occurred.  Since these substances are present in higher concentrations, they serve as substrates for continued fermentative activities with maintenance of an acid reaction in both slant and butt.

3.      Alkaline slant (red) and alkaline butt (red) or no change (orange-red) butt: No carbohydrate fermentation has occurred.  Instead, peptones are catabolized under anaerobic or aerobic conditions, resulting in an alkaline pH due to production of ammonia.  If only aerobic degradation of peptones occurs, the alkaline reaction is evidenced only on the slant surface.  If there is aerobic and anaerobic utilization of peptone, the alkaline reaction is present on the slant and the butt.

To obtain accurate results, it is essential to observe the cultures within 18 to 24 hrs following incubation.  This will ensure that the carbohydrate substrates have not been depleted and that degradation of peptones yielding alkaline end products has not taken place. 

The TSI medium also contains sodium thiosulphate, a substrate for hydrogen sulphide (H₂S) production, and ferrous sulphate for detection of this colorless end product.  Following incubation, only cultures of organisms capable of producing H₂S will show an extensive blackening in the butt because of the precipitation of the insoluble ferrous sulphide. 

Requirements

24 hr nutrient broth cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Triple Sugar-Iron Agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

 1.      Using sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of a stab and streak inoculation. One tube kept as control.

2.      Incubate for 18-24 hrs at 37^oC.

3.    3.  Examine the color of both the butt and slant of all inoculated tubes and determine the type of reaction that has taken place (acid, alkaline or none) and the carbohydrate that has been fermented in each culture.

4.     4.  Examine all cultures for the presence or absence of blackening with in the medium and determine the ability of the organism to produce H₂S.

 Observations    

Bacillus               : Acidic slant, no color change in butt, no gas and no H₂S formation.                            

 Streptococcus     : Acidic slant, acidic butt, gas produced and no H₂S formation.                                     

 Staphylococcus   : Acidic slant, acidic butt, no gas and no H₂S formation.

E.coli                   :  Acidic slant, acidic butt, gas produced and no H₂S formation.                                   

 Pseudomonas     :  Alkaline slant, no color change in butt, no gas and no H₂S formation.

 Result

 Bacillus                :A/ NC                                                                                                                         

 Streptococcus     :   A/A, G                                                                                                                          

 Staphylococcus   :   A/A

 E.coli                    :  A/A, G

 Pseudomonas      :  K/NC

Table 1. Result interpretation of Triple Sugar-Iron Agar test

Results (slant/butt)	Symbol	Interpretation
Red/yellow	K/A	Glucose fermentation only; Peptone catabolized
Yellow/yellow	A/A	Glucose and lactose and/or sucrose fermentation
Red/red	K/K	No fermentation; Peptone catabolized
Red/no color change	K/NC	No fermentation; Peptone used aerobically
Yellow/yellow with bubbles	A/A,G	Glucose and lactose and/or sucrose fermentation; Gas produced
Red/yellow with bubbles	K/A,G	Glucose fermentation only; Gas produced
Red/yellow with bubbles and black precipitate	K/A,G, H2S	Glucose fermentation only; Gas produced; H2S produced
Red/yellow with black precipitate	K/A, H2S	Glucose fermentation only; H2S produced
Yellow/yellow with black precipitate	A/A, H2S	Glucose and lactose and/or sucrose fermentation; H2S produced
No change/no change	NC/NC	No fermentation
A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

 §  Triple Sugar-Iron Agar slants

Beef extract                                        3.0 g

Yeast extract                                       3.0 g

Peptone                                               15.0 g

Proteose peptone                                 5.0 g

Lactose                                                10.0 g

Saccharose                                          10.0 g

Dextrose                                              1.0 g

Ferrous sulfate                                    0.2 g

Sodium chloride                                  5.0 g

Sodium thiosulphate                           0.3 g

Phenol red                                           0.024 g

Agar                                                    12.0 g

Distilled water                                    1 litre

pH                                                       7.4