Penicillium Microscopy

Requirements, Preparation & Observation


Introduction 


Penicillium ascomycetous fungi are microscopic organisms that are of major importance in the natural environment, in the production of foods and in the pharmaceutical industry.

The genus penicillin molecule is a well-known antibiotics drug used in medicine to fight infection from certain kinds of bacteria.  Others of this species are used in the food making industry specifically in the production of cheese. 



Its microscopy can make the organism pass for an interesting life form to study with the aid of the microscope; specimens of the organism are readily available in one's own refrigerator which can be a fruitful search area.  

Microorganisms like bacteria, yeasts, and molds, can be found in even the regularly cleaned refrigerators, however, the most fruitful search for Penicillium is one conducted in a refrigerator that has been left without cleaning for a period of 2 months or more.  


Characteristics of Penicillium


The organism is a saprophytic fungus are mostly present in the soil, in the air, and in decaying organic matter and is most commonly referred to as the green or blue mold. 

Penicillium species are widely notable for their numerous and closely packed brush-like structures that produce spores which are called penicilli (sing.: penicillus). They possess simple or branching  structures that are slightly elongated and end in clusters of flask-shapes known as phialides and are called conidiophores.

The spores also are known as conidia. They are manufactured or produced in dry chains and they emanate from the tips of the phialides. The oldest spores occupy the apex of the phialides while the youngest spores are found at the base of the phialides.

One important feature in the identification of Penicillium species is its branching as can be seen in some species like P. glabrum which are unbranched and they just bear one cluster of phialides occupying the top of the stipe.

Its vegetative body is known as mycelial and is to a great degree branched with septate hyphae, which is composed of thin-walled cells made up of one or more nuclei. Each septum possesses a central pore necessary for the maintenance of cytoplasmic continuity.

In some of the vegetative body, mycelia grow much deeper into the substratum in order to absorb food nutrients while others persist on the substrate to form a mycelial felt. Food in penicillium is stored in the form of oil globules.



Reproduction in Penicillium


Penicillium has the ability to reproduce by means of vegetative, asexual and sexual reproductive methods.


1. Vegetative reproduction:


This occurs by the splitting of vegetative mycelium into two or more parts with each part growing individually just like the parent mycelium.


2. Asexual Reproduction:


This occurs by the formation of fixed or immovable asexual spores, known as conidiophores.


  • Conidiophores:  The vegetative body or mycelium gives rise to simple and long conidiophores which branch at about two thirds of the way to the apex, in a fashion that's typical of a broom. The conidiophores branches terminate in a cluster of conidiogenous cells known as the phialides that give off chains of conidia at their apex.   
  • Conidia: The conidia appear as tiny, spore-like structures with a single nucleus. The walls of the spore are pigmented and are as well differentiated into two layers, with the outer thick, ornamented layer known as the exine; while the inner smooth and thin layer is known as the intine. The conidia are expelled from the conidiophores and become airborne to land on a favorable substratum for germination by the formation of a germ tube which elongates and becomes septates, giving rise to new hyphae.


3.  Sexual Reproduction: 


The complete state of the organism, Penicillium is assigned to two different genera which are the Eupenicillium and Talaromyces. The male sex organs of Penicillium are called antheridia, while the female sex organs are known as ascogonia.


  • Fertilization: fertilization occurs when the apex of the antheridium touches the walls of the ascogonium and subsequently the regions of touch between the two organs dissolve to give rise to a pore. The gametangia’s protoplast touches each organ through this very pore. The nuclei of the antheridial and ascogonial form a number of pairs with each pair known as a dikaryon.

4.  Occurrence:


The most common and widely notable occurrence of the species of Penicillium in food has posed a particular challenge. Some species of the organism produce toxic substances that may have the capability of rendering food products inedible or even harmful to health.

It is a good hygienic practice to do away with foods manifesting the development or appearance of any kind of mold. However, on the other side of the same coin, certain Penicillium species have been proven to be of benefit to humans.

Cheese products are made ripe with species of the organism and are certified safe for human consumption. Also, the drug penicillin is manufactured from P. chrysogenum, a mold commonly present in most homes.



Penicillium Microscopy


USING THE COMPOUND MICROSCOPE


Requirements


  • A carrot showing tufted masses of white and blue-green fungi.
  • Microscope slide
  • Compound microscope with power supply and illuminants.
  • Hematoxylin stain
  • Clean coverslip 
  • Forceps
  • Oil for immersion

Procedure


General Tip:  Every careful search in the home refrigerator will turn up a minimum of one piece of moldy food produce:


Samples of the mold used in this exercise were obtained from the food with forceps and they were placed on a clean slide of the microscope. Freshly prepared hematoxylin stain was added to the slide in three drops and made to soak right into the samples of the mold.  

The clean cover slip was gently placed on the setup and bubbles of air as many as possible were passed out of the setup gently.

The slide was studied with a compound microscope. The field of view offering the most detail possible with the compound microscope is usually a 1000X magnification.

With the use of a 400X, position the microscope slide on the stage such that the area of interest becomes clearly visible. With the stage well racked down, add a small drop of immersion oil on the top of the coverslip and also on any area of interest, then put the immersion lens in place.

  1. Turn the revolving turret of the microscope so that the lowest power objective lens is clicked into the 40X position.
  2. Place the microscope slide in position and keep it in place firmly while gripped with the clips.
  3. Look through the microscope’s eyepiece and then move the focus knob carefully for the image to come into clear focus.
  4. Slightly adjust the microscope’s condenser and amount of illumination for optimum light intensity.
  5. Move the microscope slide around until the sample is in the center of the field of view. High illumination from a small angle to the top of the sample is also helpful. 
  6. With the focus knob, carefully place the image into clear focus and also readjust the condenser and amount of illumination for a clear image.
  7. Once the image of the sample comes into clear focus with the 10X power objective, you can then switch to the net higher or lower objective to zoom in or out of the image for clarity. You can at this point place the objective into different planes for better observation of its dimensions.
  8. When you’re done with the viewing, lower the stage, then click the objective into the low lens power and take out the slide.


Observation


Starting with the objective at a 40X total magnification, the hematoxylin stained fungi sample appeared like tortuous masses of thin stalks which are known to be the hyphae. Sometimes these masses terminate in complex structures that prompted closer and more careful inspection.

Moving up the objective to a 100X total magnification, these complex structures appeared much clearer and can easily be likened to the tentacles of a sea anemone or squashed flowers in appearance.

Further at a 400X total magnification of the objective, some individual spherical structures are known to be the conidia could be seen. The "squashed flower-like" structure known to be conidiophore was difficult to be brought into focus initially but became visible on moving up the objective and thanks to its 3-dimensional appearance as compared to the rest of the hyphae which were only in 2 dimensions.


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