Effects of Lowenstein- Jensen Medium on Mycobacterium Tuberculosis Growth

Okoye L. A., Owuama C. I. and Ewanshia J. U.

Department of Microbiology MAU

All Corresponding to: ohakpolamugwo Nwabueze C . Email:ciowuama@gmail.com, ewansihajoel@mau.edu.ng

ABSTRACT

Mycobacterium tuberculosis (MTB) growth rate on Lowenstein- Jensen (L-J) medium takes 21 to 60 days. This study modified the L-J medium to achieve a faster growth rate. Both conventional and modified L-J powder containing potato flour (L-J modification 1) were used at the preliminary improvement stage. It was observed that the higher the concentration of the egg, the faster the smears from the L-J slants showed acid fast bacilli (AFB) positive results. Modified L-J slants containing 70% and 75% egg yielded MTB growth in 5 -8 days. The modified L-J 2 medium contained 70% egg and 0.5 g/Lof sodium citrate, without the potato flour present in the conventional L-J medium. Because MTB growth occurred in 5 days in the modified L-J 2 medium, it would be possible for both MTB isolation and antibiotic sensitivity test to be completed in less than 14 days. Thus the modified L-J medium 2 is recommended for quicker isolation and confirmation of the MTB cases. Conventional L-J contained low quantity of egg than the modified L-J 1 and 2. It also contained higher potatoe flour than modified L-J. Modified L-J contained no potatoe flour but contained 70ml of fresh egg.

Keywords: Tuberculosis, TBAgMPT64, Mycobacterium, Acid Fast bacilli, Lowenstein-Jensen Medium.

INTRODUCTION

Background The history of tuberculosis (TB)infection dates along with the human history, and TB probably have been existing over 70,000 years ago but remained sporadic to date (Luiser, 2012). Its infection becomes complicated when co-infected with Human Immunodeficiency Virus (HIV) (Singh, 2019).

Concealment of Mycobacterium tuberculosis (MTB) infection, drunkenness or alcoholic consumption, carefree attitudes towards its means of transmission, and superstition have all made the spread of TB rapid (Kaufmann et al., 2013). TB-HIV coinfection, weakened potency of Bacillus Calmette Guerin (BCG) vaccine and massive migration from rural to urban cities and across continental boundaries led to emergency of multidrug resistant TB (MDR-TB) Kaufmann (2013). Transmission of TB is via coughing, sneezing, shouting, crying, laughing, smoking cigarette or drinking from the same bowel with the TB infected patient. Poor or non-notification of TB infection with inadequate awareness also aids spread of TB, Some of them may be drug resistant, multi-drug resistant or extensive drug resistant tuberculosis (Sanchez et al, 2012). Tuberculosis should be treated promptly with respect and dignity the patient deserves (Philipp et al. 2011).

The greatest challenge to TB infection is isolation due to its slowness in growing in L-J medium that takes 21 to 60 days, and has identification problem also (Padmawali et al, 2016; Muril et al, 2022 and Sagar, 2022). The disease is spreading very fast beyond the medical control measures against its infection (Adegboyaga et al. 2014).

To overcome its isolation challenge, a modification of TB growth medium will be considered in this research to improve the growth rate and identification process.. The aim of this research is therefore to improve L-J medium to facilitatefaster growth of MTB, identify the Mycobacterium tuberculosis complex (MTBC) and determine the rifampicin resistant MTB isolates.

MATERIALS AND METHODS

Sample Collection

Five hundred sputum samples were collected from 265 (53%) males and 235 (47%) females who were screened for Mycobacterium tuberculosis (MTB). The Ziel-Neelsen's (ZN) staining method was used to screen for MTB from the sputum samples in this study.

Culturing of the Sputum Samples

Conventional L-J medium (37.24g) was weighed, dissolved in 600mL of distilled water containing 12 mL of glycerol. 70ml of fresh whole egg was aseptically washed, carefully cracked into 1-litre measuring cylinder, mixed carefully and blended aseptically in a sterile blender. The L0 J medium was autoclaved at 121 C, 15psi for 15 minutes. 0 When it cooled to about 50 C, the blended 70ml of fresh whole egg was added and mixed by swirling. 10ml each were then dispensed into sterile McCartney bottles, slanted, 0 and inspissated at 80 C for one hour daily for 3 consecutive days.

Purification and Liquefaction of the Sputum Samples

Freshly prepared 4% sodium hydroxide (NaOH) was prepared with sterile distilled water (SDW). Equal volume 0 of sputum sample was mixed with NaOH, incubated at 37 C for 20 minutes. The sputum-NaOH solution was mixed by turning the container upside down three times after every 5 minutes for 20 minutes. The samples were spun at 3000 rpm for 20 minutes in a Bio-Safety cabinet. The supernatant was carefully discarded into a solution of 5% Lysol or 10% freshly prepared sodium hypochlorite while the deposit was suspended in sterile distilled water (SDW).

Modification of L-J Medium

Effects of Lowenstein- Jensen Medium

Modified L-J 1 powder included potato flour while modified L-J 2 excluded potato flour and included sodium citrate. The modified L-J medium that was formulated contained asparagine 7.5g/L; dipotassium phosphate (KH PO ) 5.0g/L; magnesium citrate 1.2g/L; magnesium 2 4 sulphate 0.5g/L; malachite green 0.3 g/L, sodium citrate 0.4g/Las well as 7.4 mL/Lof glycerol, 292.6 mL/Lof SDW, 700mL/L of fresh, 4-day old whole egg and 690mg/L of amoxicillin, 690 mg/L of difflucan, 690mg/L of gentamycin, 690 mg/L of azithromycin, 690 mg/L of nalidixic acid and 690 mg/L of ciprofloxacin. During the preparation, the modified L-J medium was dissolved in SDW, glycerol was added into the solution and they were 0 sterilized at 121 C, 15 psi pressure for 15 minutes. When it 0 cooled to 50 C, the fresh 4-day old blended whole egg was added and mixed by gentle swirling without forming air bubbles. With sterile McCartney bottles, slants were made 0 and inspissated at 80 C for 1 hour for 3 consecutive days.

Inoculation of the Slants

With a sterile micropipette, 10µL of purified, extracted Mycobacteria was inoculated unto the slants. Four hundred and sixty-three purified sputum samples were inoculated on the modified L-J and 50 of the same were inoculated into the conventional L-J to serve as controls. They were incubated 0 at 37 C aerobically and monitored every 24 hours

Identification of the Isolated MTB

With sterile wireloop a colony or two was picked, emulsified on a clean microscope slide, stained with ZN method and examined with ×100 objectives and immersion oil.

TBAgMPT tests were carried out on grown MTB to 64 confirm MTB complex while GeneXpert was used to confirm rifampicin resistant MTB in sputum samples. Drug sensitivity tests of the MTB isolates was done with modified L-J medium.

Procedures:

TBAgMPT with 100ml reagent buffer pipetted into a 64: test tube, one or two colonies of MTB was emulsified and 100µl of the mycobacterial suspension siphoned and pipetted into the TBAgMPT testing capsule and left for 64 0 0 20min at 25 C - 30 C.

GeneXpert:

10ml of Xpert buffer was added into the cup containing the sputum and lightly colored the cup. The cup was vigorously shaken with back and forth movement for 10 0 0 times and left at 25 C - 30 C for 20min. Additional 10ml of the buffer was added and shaken repeatedly where the sputum was not completely liquefied. The liquefied sputum was siphoned to 2ml and placed into the GeneXpert cartridge. The cartridge was inserted with the GeneXpert machine. The machine was switched on to run for 1hr, 50min. At completion, the machine stopped automatically and the results were recorded.

RESULTS

Modified L-J 1 was the preliminary medium formed with potato flour. MTB grew on both improved L-J medium but grew faster on the improved L-J 2 slant without potato flour

Key:

L-J = Lowenstein-Jensen Five hundred sputum samples were collected. The growth time (days) plotted against the concentration of egg (in percentage) is represented in Figure 1.

Fig 1: Change in Mycobacterial growth period (days) with egg concentration (%)

The growth of Mycobacteria on 70% egg modified L-J 1 medium containing potato flour appeared on day 17 but that of the modified L-J 2 medium without potato flour th appeared on the 5 day. From the growth curve (Fig 1), the lowest concentration of egg (3%) grew MTB in 76 days on modified L-J 1 medium while the growth of MTB at the same concentration in modified L-J 2 medium grew MTB th on the 68 day.

Identification of Mycobacterial Isolates

Table 1 indicates the results obtained from 500 sputum samples . From the ZN results, all the 463 MTB isolates were ZN positive, TBAgMPT test detected 443 MTBC 64 positive and 15 negative while GeneXpert result indicated 427 MTB with 423 MTB/Rifampicin sensitive (RS), 2 MTB/ Rifampicin resistant (RR) and 2 MTB/Rifampicin indeterminate (RI). 37 patients samples did not show ZN positive, no growth of MTB on the L-J media and no positive with GeneXpert or TBAgMPT tests. Thus, they 64 had no TB infection.

Keys

No Exam = Total no of Samples worked on. MTB isolate = Total no of MTB isolated from the L-J culture L-J = MTB isolated from Conventional L-J Medium (+) = Positive L-J 1 = MTB isolated from mod. L-J 1 = Negative L-J 2 = MTB isolated from mod. L-J (2) L-J = Lowenstein –Jensen medium No MTB = Samples that grew no MTB, nor GeneXpert positive Z N = Z i e l - N e e l s e n MPT64= TBAgMPT64

DISCUSSION

The conventional L-J medium preparation was adopted from National Tuberculosis and Leprosy control program (NTBLCP2013), Laboratory manual. One challenge facing isolation of MTB was the growth duration on artificial media that takes between 21 -60 days. This research worked on modifying Lowenstein –Jensen medium for faster mycobacterial growth

The modified L-J medium contained potato flour in the first attempts of the modification. At the lowest concentration of egg (3%) MTB grew in the medium in 78 days while in at egg concentrations of 70% and 75% MTB grew in 17 days. The presence of sodium citrate, high concentration of egg, purified and liquefied sputum contribute immensely to the earlier isolation of MTB with modified L-J (Rath et al 2013; Sophie et al, 2019). The improved L-J was used to carry out drug sensitivity test of th the isolated MTB by proportion method, it grew on the 5 day unlike others that took longer days (Rajani et al, 2013). Because MTB was isolated in 5 days from modified L-J, both culture and sensitivity of patients' samples can be achieved in 14 days. Mycobacterium tuberculosis grows on potato L-J medium but it does better on modified L-J medium without potato flour.

The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-MTBC using TBAgMPT test 64 kit is consistent with the reports of Ryan, Barha and Putra (Ryan et al 2014; Barha and Putra, 2019). In addition the purification of sputum samples with 4% freshly prepared sodium hydroxide (NaOH), 69 µL/100 mL ciprofloxacin, 69 µL/100 mLgentamycin, 69 µL/100 mLazithromycin, 69 µL/100 mL nalidixic acid, 69 µL/100 mL amoxiallin, 69 µL/100 mL difflucan were added to the modified L-J to ward off contaminants (Burns and Rolain, 2014; Clair et al, 2016). MTB/Rifampicin resistant tests to find resistant mycobacteria were carried out with GeneXpert method (Vivek et al, 2020). This work adopted the purification of sputum samples prescribed by Giovanni et al, (2013) Catherine and her team used Vitamin C to boost the effects of anti-TB in their work (Catherine et al, 2013; Gaglani et al,2023).

Anti-TB drug sensitivity approaches used were directly and proportional methods. They align with Rajani et al.(2013) methods. The results were consistent and were achieved within 10-14 days from the first inoculation of MTB.

To identify the MTB growth early from the culture, use can be made of magnifying lens. This aligns with the work of Ramzi and Dedier.(2014).

Conclusion and Suggestion

We concluded that this research demonstrated early isolation, differentiation and identification of Mycrobacteria isolated from this work.

The modified L-J was found very helpful in achieving this success. Therefore, these new approaches are recommended for the routine TB diagnosis as well as in TB research programmes

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