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Year : 2019  |  Volume : 16  |  Issue : 1  |  Page : 8-10

The importance of microscopic diagnosis of respiratory infections

1 Department of Microbiology, Apollo Speciality Hospitals, Chennai, Tamil Nadu, India
2 Department of Pulmonary and Critical Care Medicine, Primus Super Speciality Hospital, New Delhi, India

Date of Web Publication11-Mar-2019

Correspondence Address:
Isabella Princess
Apollo Speciality Hospitals, Vanagaram, Chennai - 600 095, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_88_18

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The need for mastering microscopic diagnosis arises in all laboratory specialties, especially when working in resource-poor community laboratories which lack newer diagnostic instruments. Certain clues from microscopic appearance should be carefully analyzed to clinch diagnosis and to not miss out these findings. In addition, if turnaround time is reduced, there is better patient outcome and satisfaction from the clinical colleagues.

Keywords: Differential stains, fluorescent microscopy, microscopy, wet mount

How to cite this article:
Princess I, Vadala R. The importance of microscopic diagnosis of respiratory infections. Apollo Med 2019;16:8-10

How to cite this URL:
Princess I, Vadala R. The importance of microscopic diagnosis of respiratory infections. Apollo Med [serial online] 2019 [cited 2022 Dec 1];16:8-10. Available from: https://apollomedicine.org/text.asp?2019/16/1/8/253877

  Introduction Top

The major obligation of any laboratory is to provide accurate diagnosis within the shortest time frame possible. This becomes more stringent while processing samples from critically ill patients and patients with an inconclusive or atypical clinical presentation. The current state of microbiological diagnosis is centered around rapid automated diagnostic procedures with a major intention of reducing the turnaround time. However, in resource-poor settings and in low-income countries, procuring automated instruments and establishing state of art laboratory facilities is an ongoing endless challenge. Many reasons such as financial crisis and poor resources contribute to this constraint. In such cases, the only option available is mastering and using conventional procedures to the best ability for making an appropriate diagnosis.

  Importance of Microscopic Examination in the Detection of Microorganisms Top

A simple light microscope forms the cornerstone of any laboratory-based diagnostic specialty. It has not failed to lose its importance even after the advent of newer diagnostic tools and machines. Unanticipated findings on microscopy are possible even on nonsuspicion of the identified pathogen. For example, an immunocompromised patient presenting with signs of atypical pneumonia can harbor any kind of microorganism such as fungi, parasite, and atypical mycobacteria, rather than the usual bacterial pathogens causing pneumonia. A simple wet mount examination under the microscope can reveal parasites and fungal elements which can be missed on bacteriological stain, culture, and molecular diagnostic methods for viruses.[1] In any patient admitted with a diagnosis of community-acquired pneumonia, tracheal aspirate or sputum is commonly sent for bacterial stain (Gram stain) and culture; whereas, fungal mount is unusually preferred. On suspicion of tuberculosis, acid-fast stain and culture are prescribed. In high-throughput and high-resource laboratories, molecular diagnostic tests are done such as Gene Xpert, polymerase chain reaction, and line probe assay.[2]

  Microscopic Examination of Clinical Samples Top

Tracheal aspirates, sputum, and bronchoalveolar lavage should always be examined under wet mount as helminthic larvae and fungal elements can be identified at the earliest. These findings can be incidental because most of these pathogens masquerade other bacterial infections such as tuberculosis, melioidosis, and nocardiosis. When a patient from the community presents to an outpatient department or emergency with pneumonia, one usually does not anticipate parasitic infection or fungal pneumonia justifying the choice of performing bacterial stain and culture. However, if symptoms worsen or persist, other pathogens are considered, and further extensive laboratory tests are done. To eliminate the risk of misdiagnosis in the first attempt, one can perform a “package” of three or more microscopic tests to detect all types of pathogens on day 1 and within 1 h of patient admission. Therefore, rapidity of molecular tests can be replaced by equally rapid and sensitive microscopic examination done by experienced personnel. The usefulness of various microscopic diagnostic methods is elicited hereunder.

Wet mount examination

Being the easiest microscopic examination to perform and interpret, wet mount examination plays a pivotal role in easy identification of organisms within few minutes. Commonly identified respiratory pathogens on wet mount of sputum, bronchoalveolar lavage, and tracheal secretions are septate hyphae of Aspergillus species and other molds, cysts of Pneumocystis jirovecii, larvae of Strongyloides and Ascaris lumbricoides, and eggs of Paragonimus westermani and Schistosoma species.[3],[4] Wet mount does not play a major role in the diagnosis of bacterial pathogens; however, careful examination can reveal filamentous Nocardia species from clinical samples. On suspicion of pulmonary cryptococcosis, India ink or Nigrosin can be added to appreciate the capsulated yeast by its negative-staining property.

On suspicion of fungal pneumonia and subjected to availability of fluorescent microscope, Calcofluor-white dye is used to identify fungal hyphae on wet mount. Calcofluor is a fluorochrome which easily binds to polysaccharides on fungal cell wall and appears as bright blue fluorescence under the microscope. It is very sensitive and makes fungal elements easily identifiable due to the brilliant fluorescence.[5] The only disadvantage is the mandatory requirement for fluorescent microscope which might not be available in all small-scale laboratories.

After the growth of fungi on culture, lactophenol cotton blue stain is used to characterize the morphology of fungi to make a final diagnosis of the genus and species.

Differential staining methods

The most common differential stain used in the diagnosis of bacteria is Gram stain. The morphology and arrangement of bacteria reveal a lot of details on possible organisms even before growth appears on culture. The most common descriptions of bacteria and arrangement are described in [Table 1].
Table 1: Morphology and arrangement of bacteria on gram stain

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There are other instances when Gram-stain examination is useful such as detection of yeast and yeast-like fungi. Fungal hyphae, helminthic eggs, and larvae can also be detected on Gram stain on high-power objective.[6]

The other commonly used differential staining procedure is acid-fast stain or Ziehl–Neelsen stain done for the diagnosis of mycobacteria. Acid-fast bacilli could also belong to the atypical group of mycobacteria wherein culture and molecular diagnosis clarifies the doubt. Modifications which can be done are acidfast stain using 5% H2 SO4 for detecting Mycobacterium leprae and Kinyon's modification using 1% H2 SO4 for detecting Nocardia species as well as coccidian parasites from clinical samples. Cold method of acid-fast stain is also deployed for the diagnosis of  Brucella More Details species.[7]

Fluorescent staining

It is mandatory that the laboratory has fluorescent microscope to perform and interpret fluorescent staining procedures. Commonly used fluorescent stains are Auramine O for the diagnosis of mycobacteria; Calcofluor-white as described above detects fungal elements from clinical samples.

Special staining procedures

When specific emphasis is given by clinicians suspecting particular conditions such as diphtheria and to confirm pathogens from culture, some special stains are used.[8] The commonly used special stains for the detection of microorganisms are:

  • Albert stain – Differential stain used to demonstrate metachromatic granules of Corynebacterium diphtheriae
  • Endospore staining (Schaeffer–Fulton Method) – Detection of endospores on bacterial culture growth
  • Flagella staining – Silver impregnation using silver nitrate to visualize flagella of bacteria
  • Silver nitrate staining – Diagnosis of spirochetes by silver impregnation[9]
  • Negative stain/capsule stain – Quellung reaction for Streptococcus pneumoniae.

  Conclusion Top

Microbiological diagnosis has evolved to a great extent over the centuries to reach the pinnacle of successful rapid diagnosis of devastating microorganisms. However, in most of the low- and middle-income countries and in rural health-care facilities, diagnostics is purely based on microscopy and bacterial culture. Certain laboratories are still unable to standardize or procure kits for performing next-generation diagnostic techniques. In these settings, mastering the available techniques is the best option. Careful scanning of clinical samples and knowledge on pathogen identification by direct smear examination would help make best diagnosis possible in these settings. One has to keep an open mind to look for unusual microorganisms due to change in disease pattern, in the recent past due to rise in immunocompromised patients.

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Conflicts of interest

There are no conflicts of interest.

  References Top

Archibald LK. Rapid Diagnostic Testing of Infectious Diseases. Medscape Infectious Diseases; 2011. Available from: http://www.medscape.com. [Last accessed on 2018 Oct 03].  Back to cited text no. 1
Baron EJ. Rapid identification of bacteria and yeast: Summary of a national committee for clinical laboratory standards proposed guideline. Clin Infect Dis 2001;33:220-5.  Back to cited text no. 2
McClenny N. Laboratory detection and identification of Aspergillus species by microscopic observation and culture: The traditional approach. Med Mycol 2005;43 Suppl 1:S125-8.  Back to cited text no. 3
Kunst H, Mack D, Kon OM, Banerjee AK, Chiodini P, Grant A, et al. Parasitic infections of the lung: A guide for the respiratory physician. Thora×2011;66:528-36.  Back to cited text no. 4
Rasconi S, Jobard M, Jouve L, Sime-Ngando T. Use of calcofluor white for detection, identification, and quantification of phytoplanktonic fungal parasites. Appl Environ Microbiol 2009;75:2545-53.  Back to cited text no. 5
Mohan SK. Beyond bacteria: Interpreting fungal elements in the gram stain. Clin Microbiol Newsl 2004;26:108-12.  Back to cited text no. 6
Joshi PA, Kulkarni RD, Powar RM. Modified cold Z-N staining for presumptive identification of Brucella. Indian J Med Res 2005;121:108-10.  Back to cited text no. 7
Prakash BS. Microbes in Practice, IK International, New Delhi, 2014:139-55.  Back to cited text no. 8
Garvey W. Silver impregnation techniques to identify spirochetes and other bacteria. J Histotechnol 1996;19:203-9.  Back to cited text no. 9


  [Table 1]


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